U.S. patent number 8,105,813 [Application Number 11/480,188] was granted by the patent office on 2012-01-31 for materials and methods for the generation of fully 2'-modified nucleic acid transcripts.
This patent grant is currently assigned to Archemix Corp.. Invention is credited to John L. Diener, Anthony Dominic Keefe, Kristin Thompson, Chunhua Wang, Shuhao Zhu.
United States Patent |
8,105,813 |
Diener , et al. |
January 31, 2012 |
Materials and methods for the generation of fully 2'-modified
nucleic acid transcripts
Abstract
Materials and methods are provided for producing aptamer
therapeutics having fully modified nucleotide triphosphates
incorporated into their sequence.
Inventors: |
Diener; John L. (Cambridge,
MA), Keefe; Anthony Dominic (Cambridge, MA), Thompson;
Kristin (Arlington, MA), Wang; Chunhua (Acton, MA),
Zhu; Shuhao (Lincoln, MA) |
Assignee: |
Archemix Corp. (Cambridge,
MA)
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Family
ID: |
37605059 |
Appl.
No.: |
11/480,188 |
Filed: |
June 30, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070117112 A1 |
May 24, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60696292 |
Jun 30, 2005 |
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Current U.S.
Class: |
435/194; 435/183;
435/69.1 |
Current CPC
Class: |
C12Q
1/6846 (20130101); C12N 15/1048 (20130101); C12Q
1/6865 (20130101); C12N 15/115 (20130101); C12N
9/1247 (20130101); C12Q 1/6846 (20130101); C12Q
2525/117 (20130101); C12Q 2521/119 (20130101); G01N
2333/9125 (20130101); C12N 2310/16 (20130101); C12N
2330/30 (20130101); C12N 2310/321 (20130101); C12N
2310/321 (20130101); C12N 2310/3521 (20130101) |
Current International
Class: |
C12N
9/12 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 91/19813 |
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Dec 1991 |
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WO |
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WO 92/07065 |
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Apr 1992 |
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WO |
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WO 98/18480 |
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May 1998 |
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WO |
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Primary Examiner: Hutson; Richard
Attorney, Agent or Firm: Mintz Levin Cohn Ferris Glovsky and
Popeo, P.C. Elrifi; Ivor R.
Parent Case Text
REFERENCE TO RELATED APPLICATIONS
This non-provisional patent application claims priority under 35
U.S.C. .sctn.119(e) to the following provisional application: U.S.
Provisional Patent Application Ser. No. 60/696,292 filed on Jun.
30, 2005 which is herein incorporated by reference in its entirety.
The invention relates generally to the field of nucleic acids and
more particularly to aptamers.
Claims
What is claimed is:
1. An isolated T7 RNA polymerase comprising mutations at amino acid
positions 639 and 784 relative to the wild-type T7 RNA polymerase
of SEQ ID NO: 121, wherein the amino acid at position 639 is
changed to a leucine, and the amino acid at position 784 is changed
to an alanine.
2. The isolated T7 RNA polymerase of claim 1, further comprising a
mutation at amino acid position 266 relative to the wild-type T7
RNA polymerase of SEQ ID NO: 121, wherein the amino acid at
position 266 is changed to a leucine.
3. The isolated T7 RNA polymerase of claim 1, further comprising a
mutation at amino acid position 378 relative to the wild-type T7
RNA polymerase of SEQ ID NO: 121, wherein the amino acid at
position 378 is changed to an arginine.
4. The isolated T7 RNA polymerase of claim 1, wherein the mutations
increase the transcriptional yield of nucleic acids comprising
2'-OMe modifications by the polymerase in a transcription reaction
comprising only 2'-OMe nucleotide triphosphates.
5. The isolated T7 RNA polymerase of claim 4, wherein the increase
in transcriptional yield is relative to the wild-type T7 RNA
polymerase of SEQ ID NO: 121 when transcription is carried out
under identical transcription conditions.
6. The isolated T7 RNA polymerase of claim 1, wherein the mutations
decrease discrimination against 2'-OMe nucleotide
triphosphates.
7. The isolated T7 RNA polymerase of claim 6, wherein the decreased
discrimination against 2'-OMe nucleotide triphosphates is relative
to the wild-type T7 RNA polymerase of SEQ ID NO: 121.
8. An isolated polypeptide comprising the amino acid sequence of
SEQ ID NO: 1.
9. A kit comprising a container comprising the isolated T7 RNA
polymerase of claim 1 or the isolated polypeptide of claim 8.
10. The isolated T7 RNA polymerase of claim 1, further comprising
mutations at amino acid positions 266 and 378 relative to the
wild-type T7 RNA polymerase of SEQ ID NO: 121, wherein the amino
acid at position 266 is changed to a leucine, and the amino acid at
position 378 is changed to an arginine.
11. An isolated T7 RNA polymerase comprising mutations at amino
acid positions 639 and 784 relative to the wild-type T7 RNA
polymerase of SEQ ID NO: 121, wherein the amino acid at position
639 is changed to a leucine, and the amino acid at position 784 is
changed to an alanine, and wherein the mutations increase the
transcriptional yield of nucleic acids comprising 2'-OMe
modifications by the polymerase.
Description
FIELD OF INVENTION
The invention relates to materials and methods for transcribing
nucleic acids, particularly modified enzymes and materials and
methods for using the modified enzymes in template directed
polymerization to increase the incorporation of modified
nucleotides into nucleic acids, particularly aptamers.
Additionally, the invention relates to methods and materials for
selecting transcription template component sequences and the use of
such component sequences in enhancing transcript yield,
particularly in enhancing transcript yield during the SELEX.TM.
method.
BACKGROUND OF THE INVENTION
An aptamer by definition is an isolated nucleic acid molecule which
binds with high specificity and affinity to some target such as a
protein through interactions other than Watson-Crick base pairing.
Although aptamers are nucleic acid based molecules, there is a
fundamental difference between aptamers and other nucleic acid
molecules such as genes and mRNA. In the latter, the nucleic acid
structure encodes information through its linear base sequence and
thus this sequence is of importance to the function of information
storage. In complete contrast, aptamer function, which is based
upon the specific binding of a target molecule, is not dependent on
a conserved linear base sequence, but rather a particular
secondary/tertiary structure. That is, aptamers are non-coding
sequences. Any coding potential that an aptamer may possess is
entirely fortuitous and plays no role whatsoever in the binding of
an aptamer to its cognate target. Thus, while it may be that
aptamers that bind to the same target, and even to the same site on
that target, share a similar linear base sequence, most do not.
Aptamers must also be differentiated from the naturally occurring
nucleic acid sequences that bind to certain proteins. These latter
sequences are naturally occurring sequences embedded within the
genome of the organism that bind to a specialized sub-group of
proteins that are involved in the transcription, translation and
transportation of naturally occurring nucleic acids, i.e., nucleic
acid binding proteins. Aptamers on the other hand are short,
isolated, non-naturally occurring nucleic acid molecules. While
aptamers can be identified that bind nucleic acid binding proteins,
in most cases such aptamers have little or no sequence identity to
the sequences recognized by the nucleic acid binding proteins in
nature. More importantly, aptamers can bind virtually any protein
(not just nucleic acid binding proteins) as well as almost any
target of interest including small molecules, carbohydrates,
peptides, etc. For most targets, even proteins, a naturally
occurring nucleic acid sequence to which it binds does not exist.
For those targets that do have such a sequence, i.e., nucleic acid
binding proteins, such sequences will differ from aptamers as a
result of the relatively low binding affinity used in nature as
compared to tightly binding aptamers.
Aptamers, like peptides generated by phage display or antibodies,
are capable of specifically binding to selected targets and
modulating the target's activity or binding interactions, e.g.,
through binding aptamers may block their target's ability to
function. As with antibodies, this functional property of specific
binding to a target is an inherent property. Also as with
antibodies, although the skilled person may not know what precise
structural characteristics an aptamer to a target will have, the
skilled person knows how to identify, make and use such a molecule
in the absence of a precise structural definition.
Aptamers also are analogous to small molecule therapeutics in that
a single structural change, however seemingly minor, can
dramatically effect (by several orders of magnitude) the binding
and/or other activity (or activities) of the aptamer. On the other
hand, some structural changes will have little or no effect
whatsoever. This results from the importance of the
secondary/tertiary structure of aptamers. In other words, an
aptamer is a three dimensional structure held in a fixed
conformation that provides chemical contacts to specifically bind
its given target. Consequently: (1) some areas or particular
sequences are essential as (a) specific points of contact with
target, and/or as (b) sequences that position the molecules in
contact with the target; (2) some areas or particular sequences
have a range of variability, e.g., nucleotide X must be a
pyrimidine, or nucleotide Y must be a purine, or nucleotides X and
Y must be complementary; and (3) some areas or particular sequences
can be anything, i.e., they are essentially spacing elements, e.g.,
they could be any string of nucleotides of a given length or even
an non-nucleotide spacer such as a PEG molecule.
Discovered by an in vitro selection process from pools of random
sequence oligonucleotides, aptamers have been generated for over
130 proteins including growth factors, transcription factors,
enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15
kDa in size (20-45 nucleotides), binds its target with nanomolar to
sub-nanomolar affinity, and discriminates against closely related
targets (e.g., aptamers will typically not bind other proteins from
the same gene family). A series of structural studies have shown
that aptamers are capable of using the same types of binding
interactions (e.g., hydrogen bonding, electrostatic
complementarities, hydrophobic contacts, steric exclusion) that
drive affinity and specificity in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as
therapeutics and diagnostics including high specificity and
affinity, biological efficacy, and excellent pharmacokinetic
properties. In addition, they offer specific competitive advantages
over antibodies and other protein biologics, for example:
1) Speed and control. Aptamers are produced by an entirely in vitro
process, allowing for the rapid generation of initial leads,
including therapeutic leads. In vitro selection allows the
specificity and affinity of the aptamer to be tightly controlled
and allows the generation of leads, including leads against both
toxic and non-immunogenic targets.
2) Toxicity and Immunogenicity. Aptamers as a class have
demonstrated therapeutically acceptable toxicity or lack of
immunogenicity. Whereas the efficacy of many monoclonal antibodies
can be severely limited by immune response to antibodies
themselves, it is extremely difficult to elicit antibodies to
aptamers most likely because aptamers cannot be presented by
T-cells via the MHC and the immune response is generally trained
not to recognize nucleic acid fragments.
3) Administration. Whereas most currently approved antibody
therapeutics are administered by intravenous infusion (typically
over 2-4 hours), aptamers can be administered by subcutaneous
injection (aptamer bioavailability via subcutaneous administration
is >80% in monkey studies (Tucker et al., J. Chromatography B.
732: 203-212, 1999)). This difference is primarily due to the
comparatively low solubility and thus large volumes necessary for
most therapeutic mAbs. With good solubility (>150 mg/mL) and
comparatively low molecular weight (aptamer: 10-50 kDa; antibody:
150 kDa), a weekly dose of aptamer may be delivered by injection in
a volume of less than 0.5 mL. In addition, the small size of
aptamers allows them to penetrate into areas of conformational
constrictions that do not allow for antibodies or antibody
fragments to penetrate, presenting yet another advantage of
aptamer-based therapeutics or prophylaxis.
4) Scalability and cost. Therapeutic aptamers are chemically
synthesized and consequently can be readily scaled as needed to
meet production demand. Whereas difficulties in scaling production
are currently limiting the availability of some biologics and the
capital cost of a large-scale protein production plant is enormous,
a single large-scale oligonucleotide synthesizer can produce
upwards of 100 kg/year and requires a relatively modest initial
investment. The current cost of goods for aptamer synthesis at the
kilogram scale is estimated at $500/g, comparable to that for
highly optimized antibodies. Continuing improvements in process
development are expected to lower the cost of goods to <$100/g
in five years.
5) Stability. Therapeutic aptamers are chemically robust. They are
intrinsically adapted to regain activity following exposure to
factors such as heat and denaturants and can be stored for extended
periods (>1 yr) at room temperature as lyophilized powders. In
contrast, antibodies must be stored refrigerated.
In addition to the intrinsic stability of aptamers, modified
nucleotides (e.g., 2'-modified nucleotides) which are inexpensive,
non-toxic, and which can increase resistance to enzymatic,
chemical, thermal, and physical degradation, can be incorporated
during SELEX.TM. method as described in U.S. patent application
Ser. No. 10/729,851 filed Dec. 3, 2002, and U.S. patent application
Ser. No. 10/873,856, filed Jun. 21, 2004. While incorporation of
modified nucleotides during SELEX.TM. process is oftentimes
preferable to post-SELEX.TM. modification due to potential loss of
binding affinity and activity that can occur post-SELEX.TM.
selection, the incorporation of modified nucleotides, e.g.
2'-O-methyl nucleotides ("2-OMe"), during the SELEX.TM. process has
been historically difficult because of low transcription yields.
Solution conditions and transcription mixtures are described in
U.S. patent application Ser. No. 10/729,851 filed Dec. 3, 2002, and
U.S. patent application U.S. Ser. No. 10/873,856, filed Jun. 21,
2004, which give improved transcription yields for aptamers
incorporating 2'-OMe nucleotides. However transcription yields for
fully 2'-O-methylated aptamers remain problematic.
In addition to the advantages of aptamers as therapeutic agent,
given the inexpensive nature, low toxicity, and increased nuclease
resistance conferred by the incorporation of 2'-OMe nucleotides in
aptamers, it would be beneficial to have materials and methods to
increase transcript yields of fully 2'-O-methylated aptamers to,
e.g., prolong or increase the stability of aptamer therapeutics in
vivo. The present invention provides improved materials and methods
to meet these and other needs.
SUMMARY OF THE INVENTION
The present relates to T7 RNA polymerases, which may be purified,
isolated and/or recombinant. As used herein the term isolated
encompasses polymerases of the invention when recombinantly
expressed in a cell or tissue. As used herein the term isolated
encompasses nucleic acid sequences of the invention when engineered
into a cell or tissue In one embodiment, a T7 RNA polymerase
comprising an altered amino acid at position 639 and position 784
wherein the altered amino acid at position 639 is not a
phenylalanine when the altered amino acid at position 784 is an
alanine is provided. In another embodiment, the above described T7
RNA polymerase further comprising an altered amino acid at position
378 is provided. In another embodiment, the above described T7 RNA
polymerases further comprising an altered amino acid at position
266 is provided. In a particular embodiment the altered amino acid
at position 639 is a leucine and the altered amino acid at position
784 is an alanine. In a further embodiment, the altered amino acid
at position 266 is a leucine. In a further embodiment, the altered
amino acid at position 378 is an arginine.
In preferred embodiments, the altered amino acids increase the
transcriptional yield of nucleic acids comprising 2'-OMe
modifications by the polymerase in a transcription reaction
comprising only 2'-OMe nucleotide triphosphate. In a particular
embodiment the increase in transcription yield is relative to a T7
RNA polymerase lacking the altered amino acids when transcription
is carried out for both the altered amino acid T7 RNA polymerase
and the T7 RNA polymerase lacking the altered amino acids under
identical transcription conditions. In another embodiment, the
altered amino acids decrease discrimination against 2'-OMe
nucleotide triphosphates. In a particular embodiment, the decreased
discrimination against 2'-OMe nucleotide triphosphates is relative
to a T7 RNA polymerase lacking the altered amino acids when both
polymerases are used under identical transcription conditions. In
particular embodiments of this aspect, the T7 RNA polymerase
lacking the altered amino acids is the wild type T7 RNA polymerase
comprising an amino acid at position 639 altered to a phenylalanine
and an amino acid at position 784 altered to alanine or a mutant
polymerase having the wild type amino acid sequence except that a
phenylalanine has been substituted for the tyrosine at position
639, and an alanine has been substituted for the histidine at
position 784 and an arginine residue substituted for the lysine
residue at position 378 (Y639F/H784A/K378R).
In a particular embodiment, an isolated polypeptide comprising an
amino acid selected from the group consisting of: SEQ ID NO 1, SEQ
ID NO 2, SEQ ID NO 102 and SEQ ID NO 103. In a particular
embodiment, a kit comprising a container containing a T7 RNA
polymerase of the invention is provided.
In some embodiments, a method of transcribing a single stranded
nucleic acid comprising incubating a mutant T7 RNA polymerase with
a template nucleic acid under reaction conditions sufficient to
result in transcription is provided.
In another embodiment, an isolated nucleic acid encoding a
polypeptide of the invention is provided. In a particular
embodiment a nucleic acid sequence, selected from the group
consisting of: SEQ ID NO 122, SEQ ID NO 123, SEQ ID NO 124 and SEQ
ID NO 125 is provided. In some embodiments, a vector comprising an
isolated nucleic acid sequence of the invention is provided. In a
particular embodiment, an expression vector comprising a nucleic
acid of the invention operably linked to a promoter is provided. In
another embodiment of the invention, a cell comprising the
expression vector of the invention is provided. In a particular
embodiment, a cell wherein the mutant T7 RNA polymerase of the
invention is expressed by the cell is provided. In some
embodiments, a kit comprising a container containing a nucleic acid
encoding a T7 RNA polymerase of the invention is provided.
In another embodiment, a method of transcribing a fully 2'-OMe
nucleic acid comprising the steps of a) incubating a template
nucleic acid in a reaction mixture under conditions comprising a
mutant RNA polymerase, a nucleic acid transcription template and
nucleoside triphosphates, wherein the nucleoside triphosphates are
2'OMe, and b) transcribing the transcription reaction mixture to
result in single stranded nucleic acid, wherein all of the
nucleotides of the single stranded nucleic acids are 2'-OMe
modified except that the first nucleotide of the transcripts can be
2' unmodified, is provided. In some embodiments, the first
nucleoside of the transcript may be 2'-OH guanosine. In some
embodiments of the method, the mutant RNA polymerase is a mutant T7
RNA polymerase comprising an altered amino acid at position 639 and
position 784, particularly a T7 RNA polymerase comprising an
altered amino acid at position 639 and position 784 wherein the
altered amino acid at position 639 is not a phenylalanine when the
altered amino acid at position 784 is an alanine, particularly, a
T7 RNA polymerase further comprising an altered amino acid at
position 378 and/or an altered amino acid at position 266. In a
particular embodiment the altered amino acid at position 639 is a
leucine and the altered amino acid at position 784 is an alanine in
the polymerase for use in the methods of the invention. In a
further embodiment, the altered amino acid at position 266 is a
leucine of the polymerase for use in the methods of the invention.
In a further embodiment, the altered amino acid at position 378 is
an arginine in the polymerase for use in the methods of the
invention. In a particular embodiment, an isolated polypeptide
comprising an amino acid selected from the group consisting of: SEQ
ID NO 1, SEQ ID NO 2, SEQ ID NO 102 and SEQ ID NO 103 is
provided.
In some embodiments of the method of the invention, the
transcription reaction further comprises magnesium ions. In another
embodiment, the transcription reaction further comprises manganese
ions. In another embodiment, the magnesium ions are present in the
transcription reaction at a concentration that is between 3.0 to
3.5 times greater than the manganese ions. In another embodiment,
wherein each nucleotide triphosphate is present in the
transcription reaction at a concentration of 1.0 mM, the
concentration of magnesium ions is 6.5 mM, and the concentration of
manganese ions is 2.0 mM. In another embodiment, wherein each
nucleotide triphosphate is present in the transcription reaction at
a concentration of 1.5 mM, the concentration of magnesium ions 8
mM, and the concentration of manganese ions is 2.5 mM. In another
embodiment, wherein each nucleotide triphosphate is present in the
transcription reaction at a concentration of 2.0 mM, the
concentration of magnesium ions 9.5 mM and concentration of
manganese ions is 3.0 mM.
In another embodiment, the transcription reaction further comprises
a non 2'-OMe guanosine non-triphosphate residue, particularly
wherein the non 2'-OMe guanosine non-triphosphate residue selected
from the group consisting of: guanosine monophosphate, guanosine
diphosphate, 2' flouro guanosine monophosphate, 2' flouro guanosine
diphosphate, 2'-amino guanosine monophosphate, 2'-amino guanosine
diphosphate, 2'-deoxy guanosine monophosphate, and 2'-deoxy
guanosine diphosphate. In another embodiment, the transcription
template comprises a T7 RNA polymerase promoter. In another
embodiment, the transcription reaction further comprises
polyethylene glycol. In another embodiment, the transcription
reaction comprises inorganic pyrophosphatase.
In another aspect of the invention a method for identifying
aptamers is provided. In one embodiment, a method for identifying
an aptamer, comprising: a) preparing a transcription reaction
mixture comprising a mutant polymerase of the invention, and one or
more nucleic acid transcription templates) transcribing the
transcription reaction mixture to result in a candidate mixture of
single stranded nucleic acids, wherein all but optionally one of
the nucleotides of the single stranded nucleic acids are
2'modified, c) contacting the candidate mixture with the target
molecule, d) partitioning the nucleic acids having an increased
affinity for the target molecule, relative to an affinity of the
candidate mixture, from the candidate mixture, and e) amplifying
the increased affinity nucleic acids to yield an aptamer enriched
mixture, whereby aptamers to the target molecule comprise all
2'-modified nucleotide except that the first nucleotide of the
aptamers can be 2'-unmodified are identified, is provided. In some
embodiments, the amplifying step f) comprises (i) optionally
dissociating the increased affinity nucleic acids from the target,
ii) reverse transcribing the increased affinity nucleic acids
dissociated from the nucleic acid-target complexes, iii) amplifying
the reverse transcribed increased affinity nucleic acids; and (ii)
preparing a transcription reaction mixture comprising the amplified
reverse transcribed increased affinity nucleic acids as the
transcription template and transcribing the transcription
mixture.
In some embodiments of the aptamer identification method of the
invention, the mutant RNA polymerase is a mutant T7 RNA polymerase
comprising an altered amino acid at position 639 and position 784,
particularly a T7 RNA polymerase comprising an altered amino acid
at position 639 and position 784 wherein the altered amino acid at
position 639 is not a phenylalanine when the altered amino acid at
position 784 is an alanine, particularly, a T7 RNA polymerase
further comprising an altered amino acid at position 378 and/or an
altered amino acid at position 266. In a particular embodiment the
altered amino acid at position 639 is a leucine and the altered
amino acid at position 784 is an alanine in the polymerase for use
in the methods of the invention. In a further embodiment, the
altered amino acid at position 266 is a leucine of the polymerase
for use in the methods of the invention. In a further embodiment,
the altered amino acid at position 378 is an arginine in the
polymerase for use in the methods of the invention. In a particular
embodiment, an isolated polypeptide comprising an amino acid
selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2,
SEQ ID NO 102 and SEQ ID NO 103 is used in the aptamer
identification method of the invention.
In some embodiments, the all the nucleotide triphosphates in the
transcription reaction are 2'-OMe modified. In one embodiment, the
one or more nucleic acid transcription template comprises a T7 RNA
polymerase promoter and a leader sequence immediately 3' to the T7
RNA polymerase promoter. In some embodiments of this aspect, the
method comprises repeating steps a) to e) iteratively.
In some embodiments of the aptamer identifying method of the
invention, the transcription reaction further comprises magnesium
ions. In another embodiment, the transcription reaction further
comprises manganese ions. In another embodiment, the magnesium ions
are present in the transcription reaction at a concentration that
is between 3.0 to 3.5 times greater than the manganese ions. In
another embodiment, wherein each nucleotide triphosphate is present
in the transcription reaction at a concentration of 1.0 mM, the
concentration of magnesium ions is 6.5 mM, and the concentration of
manganese ions is 2.0 mM. In another embodiment, wherein each
nucleotide triphosphate is present in the transcription reaction at
a concentration of 1.5 mM, the concentration of magnesium ions 8
mM, and the concentration of manganese ions is 2.5 mM. In another
embodiment, wherein each nucleotide triphosphate is present in the
transcription reaction at a concentration of 2.0 mM, the
concentration of magnesium ions 9.5 mM and concentration of
manganese ions is 3.0 mM.
In another embodiment of this aspect, the transcription reaction
for use in the aptamer identification method of the invention
further comprises a non 2'-OMe guanosine non-triphosphate residue,
particularly wherein the non 2'-OMe guanosine non-triphosphate
residue selected from the group consisting of: guanosine
monophosphate, guanosine diphosphate, 2' flouro guanosine
monophosphate, 2' flouro guanosine diphosphate, 2'-amino guanosine
monophosphate, 2'-amino guanosine diphosphate, 2'-deoxy guanosine
monophosphate, and 2'-deoxy guanosine diphosphate. In another
embodiment, the transcription template comprises a T7 RNA
polymerase promoter. In another embodiment, the transcription
reaction further comprises polyethylene glycol. In another
embodiment, the transcription reaction comprises inorganic
pyrophosphatase.
The present invention also relates to a method of selecting
component sequences of nucleic acid templates for directing
transcription. In one embodiment, the component sequence enhances
the transcript yield of template directed transcription. In a
particular embodiment, the invention relates to methods of
selecting leader sequences to enhance transcript yield and to the
leader sequences, nucleic acid templates comprising the leader
sequences and methods of using the leader sequences and nucleic
acid templates of the invention. The present invention also relates
to novel mutant polymerases and their use in transcription,
particularly its use to enhance transcript yield where 2'modified
nucleotides are being incorporated, more particularly where all the
nucleotides being incorporate are 2'modified, e.g. are 2'-OMe. The
present invention also relates to modified transcription reaction
conditions to enhance transcript yield. The present invention
particularly relates to pair wise and triple combinations of the
above aspects, particularly to improve transcript yield wherein in
all but the starting nucleotide of the transcripts are 2'-modified,
particularly 2'-OMe modified ("fully 2'-OMe" or "mRmY" or "MNA"
transcripts).
In one embodiment of the first aspect of the invention a method of
identifying a nucleic acid template component sequence for
enhancing transcription, comprising: a) preparing a library of
transcription template candidates, wherein the templates comprise a
promoter, a first fixed region immediately 3' to the promoter, a
degenerate region immediately 3' to the first fixed region and a
second fixed region 3' to the degenerate region; b) transcribing
the library of transcription template candidates in a transcription
reaction to give a library of transcripts; c) reverse transcribing
the transcription mixture to obtain a candidate mixture of cDNA
wherein the cDNA templates comprise a 5' and 3' terminus; d)
ligating a DNA sequence encoding the promoter to the 3' terminus of
the cDNA templates in a ligation reaction; e) amplifying the cDNA
templates to result in a library of transcription template
candidates; and f) identifying a nucleic acid sequence component
for enhancing transcription from the library of transcription
template candidates, wherein the nucleic acid sequence component
comprises a sequence derived from at least a portion of the
degenerate region, is provided. In one embodiment of this method of
the invention, step f) comprises i) cloning the library of
transcription template candidates into individual transcription
templates; ii) transcribing the individual transcription templates
in a transcription reaction to result in a yield of transcripts;
iii) assessing the transcript yield of the individual transcription
templates; and iv) identifying the nucleic acid sequence component
in a transcription template that results in a predetermined
transcript yield. In a particular embodiment of this method of the
invention, the predetermined transcript yield is a yield greater
than the transcript yield obtained in step b) by transcribing the
transcription template candidate mixture.
In another embodiment of this method of the invention step f)
comprises analyzing the base composition of the degenerate region
of the library of transcription template candidates and identifying
the nucleic acid sequence component based on the average base
composition of the transcription template candidate library.
In some embodiments of this aspect of the invention step b) of the
method further comprises treating the transcribed transcription
mixture with DNase. In further embodiments of this method of the
invention, step b) further comprises purifying the transcribed
transcription mixture by partitioning the transcribed transcription
templates away from other components of the transcription reaction.
In a particular embodiment of this method of the invention, the
purification step comprises replacing the transcription reaction
buffer by running the transcription reaction through a desalting
column.
In another embodiment of this aspect of the invention, step d) of
the method is done before step c). In another embodiment of this
aspect of the invention the method comprises repeating steps b) to
e) more than once prior to performing step f).
In a further embodiment of this aspect of the invention, the
ligation reaction is a splinted ligation reaction and the ligation
reaction comprises a nucleic acid splint and a
5'-monophosphorylated oligonucleotide encoding the promoter.
In a particular embodiment of this aspect of the invention, the
transcription reaction used in the method comprises one or more
modified nucleotide triphosphates and a mutated polymerase. In some
embodiments the modified nucleotide triphosphate is a 2'-modified
nucleotide triphosphate, particularly a 2'-OMe modified nucleotide
triphosphate. In some embodiments, the mutated polymerase is a
mutated T7 RNA polymerase. In some embodiments the transcription
reaction used in the method of the invention comprises magnesium
and manganese ions (Mn.sup.2+) and the mutated T7 RNA polymerase is
selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2,
SEQ ID NO 100 and SEQ ID NO 101.
In some embodiments of this aspect of the invention, the magnesium
ions are present in the transcription reaction at a concentration
that is between 3.0 to 3.5 times greater than the manganese ions
(Mn.sup.2+). In further embodiments of this aspect of the
invention, each nucleotide triphosphate is present in the
transcription reaction at a concentration of 1.0 mM, the
concentration of magnesium ions is 6.5 mM, and the concentration of
manganese ions is 2.0 mM. In further embodiments of this aspect of
the invention, each nucleotide triphosphate is present in the
transcription reaction at a concentration of 1.5 mM, the
concentration of magnesium ions 8 mM, and the concentration of
manganese ions is 2.5 mM. In still further embodiments of this
aspect of the invention, each nucleotide triphosphate is present in
the transcription reaction at a concentration of 2.0 mM, the
concentration of magnesium ions 9.5 mM and concentration of
manganese ions is 3.0 mM. In some embodiments of the method, the
transcription reaction further comprises a polyalkylene glycol,
particularly polyethylene glycol. In some embodiments of the
method, particularly embodiments in which fully 2'-OMe transcripts
are desired, the transcription reaction further comprises a
guanosine residue selected from the group consisting of: guanosine
monophosphate, guanosine diphosphate, 2' fluoro guanosine
monophosphate or diphosphate, 2'-amino guanosine monophosphate or
diphosphate, 2'-deoxy guanosine monophosphate or diphosphate, or
other modified nucleotides. In further embodiments, the
transcription reaction of the method of the invention comprises
inorganic pyrophosphatase. In further embodiments, the
transcription reaction of the method of the invention optionally
comprises combination from the group consisting of: buffer,
detergent (e.g., Triton X-100), polyamine (e.g., spermine or
spermidine), and reducing agent (e.g., DTT or .beta.ME). In yet
further embodiments, the transcription reaction of the method of
the invention comprises nucleotide triphosphates, magnesium ions,
manganese ions (e.g. Mn.sup.2+), polyethylene glycol, guanosine
monophosphate, inorganic pyrophosphatase, buffer, detergent,
polyamine, and DTT, and one or more oligonucleotide transcription
templates. and a T7 RNA polymerase, e.g. a mutant T7 RNA
polymerase, e.g. a mutant T7 RNA polymerase selected from the group
consisting of: SEQ ID NO 1, 2, 100 and 101
In some embodiments of the identification method of the invention,
the first fixed region of the library of transcription template
candidates consists of 2, 3, 4 or 5 guanosine residues. In some
embodiments of the invention, the degenerate region of the library
of transcription template candidates comprises at least 4, 10, 20
or 30 nucleotides.
In some embodiments of the method, the nucleic acid template
component sequence to be identified is a leader sequence. In some
embodiments, the leader sequence comprises the first fixed region
and a sequence derived from at least a portion of the degenerate
region of the library of transcription template candidates. In some
embodiments, the method of the invention further comprises
incorporating the identified leader sequence into an
oligonucleotide transcription template.
The invention also provides leaders sequence identified by the
identification method of the invention. In some embodiments, the
leader sequence of the invention comprises the nucleic acid
sequence from nucleotide 22 to nucleotide 32 in any one of the
sequences selected from the group consisting of: SEQ ID NOs 10 to
99. In some embodiments, the leader sequence of the invention
comprises the nucleic acid sequence from nucleotide 18 to
nucleotide 32 in any one of the sequences selected from the group
consisting of: SEQ ID NOs 10-99. The invention also provides an
oligonucleotide transcription template comprising a leader sequence
of the invention. In particular embodiments the invention provides
oligonucleotide transcription template is selected from the group
consisting of: SEQ ID NO 3 to 6 and SEQ ID NO 106.
In another aspect of the invention, a method for increasing
transcript yield of a nucleic acid where transcription is directed
by an oligonucleotide transcription template is provided. In some
embodiments of this aspect of the invention, the method of
increasing transcript yield comprises directing transcription with
an oligonucleotide transcription template that comprises a leader
sequence, wherein the leader sequence has been identified by the
identification method of the invention using the same nucleotide
composition and/or polymerase and/or conditions as used in the
transcription reaction for which enhancement of transcript yield is
desired.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the in vitro aptamer
selection (SELEX.TM.) process from pools of random sequence
oligonucleotides.
FIG. 2 shows a flow diagram of a Terminal Region SELEX.TM.
(TR-SELEX.TM.) method.
FIG. 3 shows a graphical analysis of the combined average
nucleotide composition of regions selected from the twenty
degenerate positions of a library of transcription template
candidates before (R0) and after (R3) TR-SELEX.TM. selection.
FIG. 4 shows the relative transcript yield quantitated from
UV-shadowing of PAGE-gel analysis for ARC2118, ARC2119, ARC2120,
and ARC2121 using the Y639F/H784A/K378R ("FAR") and
Y639L/H784A/K378R ("LAR") mutant T7 RNA polymerases with a 2'-OH
GTP spike in the transcription mixture. * indicates that the given
yields are relative to ARC2118 transcribed with the LAR mutant
polymerase, which gave the highest quantitated yield by
UV-shadow.
FIG. 5A shows the nucleic acid (SEQ ID NO 120) and FIG. 5B gives
the amino acid sequence of the wild type T7 RNA polymerase (SEQ ID
NO 121).
FIG. 6A shows the nucleic acid sequence (SEQ ID NO 122) of mutant
T7 RNA polymerase Y639L/H784A. FIG. 6B shows the nucleic acid
sequence (SEQ ID NO 123) of T7 mutant polymerase Y639L/H784A/K378R.
FIG. 6C shows the nucleic acid sequence (SEQ ID NO 124) of mutant
T7 polymerase P266L/Y639L/H784A. FIG. 6D shows the nucleic acid
sequence (SEQ ID NO 125) of mutant T7 polymerase
P266L/Y639L/H784A/K378R.
FIG. 7 shows the relative transcript yield quantitated from
UV-shadowing of PAGE-gel analysis for ARC2118 and ARC2119 using the
Y639L/H784A/K378R mutant T7 RNA polymerase with a titration of rGTP
(2'-OH GTP) in the transcription mixture. * indicates that the
given yields are relative to ARC2118 transcribed with 20 uM rGTP,
which gave the highest quantitated yield by UV-shadow.
FIG. 8 shows the relative transcript yield quantitated from
UV-shadowing of PAGE-gel analysis for ARC2119 using the
Y639L/H784A/K378R mutant T7 RNA polymerase with a varying
concentrations of 2'-OMe NTPs (A, U, C, and G), MgCl.sub.2 and
MnCl.sub.2 and no rGTP (2'-OH GTP) in the transcription mixture.
The given yields are relative to the 1 mM each 2'-OMe NTP, 6.5 mM
MgCl.sub.2, and 2 mM MnCl.sub.2 transcription condition.
FIG. 9 is a table that shows an analysis of the nucleotide
insertions, deletions and substitutions of fully 2'-OMe
transcription (100% 2'-OMe A, U, C, G) with the Y639L/H784A/K378R
mutant T7 RNA polymerase, compared to the fidelity of all RNA or
2'-OMe transcription using the Y639F/K378R mutant T7 RNA
polymerase. In the table, (1) indicates data from "Direct in Vitro
Selection of a 2'-O-Methyl Aptamer to VEGF" Burmeister et. al.,
(2005) Chemistry and Biology, 12: 25-33 where transcriptions were
done with FAR T7 mutant polymerase and (2) indicates that
transcription was done with LAR T7 mutant polymerase.
FIG. 10 is a table that shows an analysis of the percent nucleotide
composition of fully 2'-OMe transcripts (100% 2'-OMe A, T, C, G)
before and after one round of fully 2'-OMe transcription using the
Y639L/H784A/K378R mutant T7 RNA polymerase followed by DNase
treatment, reverse transcription, splinted ligation, and PCR
amplification.
FIG. 11 is a schematic of a minimized MNA anti-IgE aptamer shown in
the 5' to 3' direction having a cap on its 3'end (dark colored
ball).
FIG. 12 is a schematic of the minimized MNA anti-IgE aptamer, the
minimized MNA anti-IgE aptamer having two deoxy substitutions and
the minimized MNA anti-IgE aptamer having one deoxy substitution
and phosphorothioate substitutions, each shown in the 5' to 3'
direction and each having a cap on its 3'end (black colored
ball).
FIG. 13 is an illustration depicting various PEGylation strategies
representing standard mono-PEGylation, multiple PEGylation, and
dimerization via PEGylation.
DETAILED DESCRIPTION OF THE INVENTION
The details of one or more embodiments of the invention are set
forth in the accompanying description below. Although any methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present invention, the
preferred methods and materials are now described. Other features,
objects, and advantages of the invention will be apparent from the
description. In the specification, the singular forms also include
the plural unless the context clearly dictates otherwise. Unless
defined otherwise, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. In the case of
conflict, the present Specification will control.
The SELEX.TM. Method
The preferred method for generating an aptamer is with the process
entitled "Systematic Evolution of Ligands by Exponential
Enrichment" ("SELEX.TM.") generally depicted in FIG. 1 and also
referred to as in vitro selection. The SELEX.TM. process is a
method for the in vitro evolution of nucleic acid molecules with
highly specific binding to target molecules and is described in,
e.g., U.S. patent application Ser. No. 07/536,428, filed Jun. 11,
1990, now abandoned, U.S. Pat. No. 5,475,096 entitled "Nucleic Acid
Ligands", and U.S. Pat. No. 5,270,163 (see also WO 91/19813)
entitled "Nucleic Acid Ligands". By performing iterative cycles of
selection and amplification SELEX.TM. may be used to obtain
aptamers, also referred to herein as "nucleic acid ligands" with
any desired level of target binding affinity.
The SELEX.TM. process is based on the unique insight that nucleic
acids have sufficient capacity for forming a variety of two- and
three-dimensional structures and sufficient chemical versatility
available within their monomers to act as ligands (i.e., form
specific binding pairs) with virtually any chemical compound,
whether monomeric or polymeric. Molecules of any size or
composition can serve as targets.
The SELEX.TM. process is based on the ability to bind a target.
Aptamers obtained through the SELEX.TM. procedure will thus have
the property of target binding. Mere target binding, however
provides no information on the functional effect, if any, which may
be exerted on the target by the action of aptamer binding.
Alteration of a property of the target molecule requires the
aptamer to bind at a certain location on the target in order to
effect a change in a property of the target. In theory, the
SELEX.TM. method may result in the identification of a large number
of aptamers, where each aptamer binds at a different site on the
target. In practice, aptamer-target binding interactions often
occur at one or a relatively small number of preferred binding
sites on the target which provide stable and accessible structural
interfaces for the interaction. Furthermore, when the SELEX.TM.
method is performed on a physiological target molecule the skilled
person is generally not able to control the location of aptamer to
the target. Accordingly, the location of the aptamer binding site
on the target may or may not be at, or close to, one of potentially
several binding sites that could lead to the desired effect, or may
not have any effect on the target molecule.
Even where an aptamer, by virtue of its ability to bind the target,
is found to have an effect there is no way of predicting the
existence of that effect or of knowing in advance what the effect
will be. In performing a SELEX.TM. experiment the skilled person
can only know with any certainty that aptamers, to the extent it is
possible to obtain an aptamer against a target, will have the
property of target binding. One may perform a SELEX.TM. experiment
in the hope that some of the aptamers identified will also have an
effect on the target beyond binding to it, but this is
uncertain.
The SELEX.TM. process relies as a starting point upon a large
library or pool of single stranded oligonucleotides comprising
randomized sequences. The oligonucleotides can be modified or
unmodified DNA, RNA, or DNA/RNA hybrids. In some examples, the pool
comprises 100% degenerate or partially degenerate oligonucleotides.
In other examples, the pool comprises degenerate or partially
degenerate oligonucleotides containing at least one fixed sequence
and/or conserved sequence incorporated within randomized sequence.
In other examples, the pool comprises degenerate or partially
degenerate oligonucleotides containing at least one fixed sequence
and/or conserved sequence at its 5' and/or 3' end which may
comprise a sequence shared by all the molecules of the
oligonucleotide pool. Fixed sequences are sequences common to
oligonucleotides in the pool which are incorporated for a
preselected purpose such as, CpG motifs described further below,
hybridization sites for PCR primers, promoter sequences for RNA
polymerases (e.g., T3, T4, T7, and SP6), restriction sites, or
homopolymeric sequences, such as poly A or poly T tracts, catalytic
cores, sites for selective binding to affinity columns, leader
sequences which promote transcription, and other sequences to
facilitate cloning and/or sequencing of an oligonucleotide of
interest. Conserved sequences are sequences, other than the
previously described fixed sequences, shared by a number of
aptamers that bind to the same target.
The oligonucleotides of the pool preferably include a degenerate
sequence portion as well as fixed sequences necessary for efficient
amplification. Typically the oligonucleotides of the starting pool
contain fixed 5' and 3' terminal sequences which flank an internal
region of 30-40 random nucleotides. The degenerate nucleotides can
be produced in a number of ways including chemical synthesis and
size selection from randomly cleaved cellular nucleic acids.
Sequence variation in test nucleic acids can also be introduced or
increased by mutagenesis before or during the
selection/amplification iterations.
The degenerate sequence portion of the oligonucleotide can be of
any length and can comprise ribonucleotides and/or
deoxyribonucleotides and can include modified or non-natural
nucleotides or nucleotide analogs. See, e.g., U.S. Pat. No.
5,958,691; U.S. Pat. No. 5,660,985; U.S. Pat. No. 5,958,691; U.S.
Pat. No. 5,698,687; U.S. Pat. No. 5,817,635; U.S. Pat. No.
5,672,695, and PCT Publication WO 92/07065. Degenerate
oligonucleotides can be synthesized from phosphodiester-linked
nucleotides using solid phase oligonucleotide synthesis techniques
well known in the art. See, e.g., Froehler et al., Nucl. Acid Res.
14:5399-5467 (1986) and Froehler et al., Tet. Lett. 27:5575-5578
(1986). Random oligonucleotides can also be synthesized using
solution phase methods such as triester synthesis methods. See,
e.g., Sood et al., Nucl. Acid Res. 4:2557 (1977) and Hirose et al.,
Tet. Lett., 28:2449 (1978). Typical syntheses carried out on
automated DNA synthesis equipment yield 10.sup.16-10.sup.17
individual molecules, a number sufficient for most SELEX.TM.
experiments. Sufficiently large regions of degenerate sequence in
the sequence design increases the likelihood that each synthesized
molecule is likely to represent a unique sequence.
The starting library of oligonucleotides may be generated by
automated chemical synthesis on a DNA synthesizer. To synthesize
degenerate sequences, mixtures of all four nucleotides are added at
each nucleotide addition step during the synthesis process,
allowing for stochastic incorporation of nucleotides. As stated
above, in one embodiment, random oligonucleotides comprise entirely
degenerate sequences; however, in other embodiments, degenerate
oligonucleotides can comprise stretches of nonrandom or partially
random sequences. Partially random sequences can be created by
adding the four nucleotides in different molar ratios at each
addition step.
In those instances where an RNA library is to be used as the
starting library it is typically generated by synthesizing a DNA
library, optionally PCR amplifying, then transcribing the DNA
library in vitro using T7 RNA polymerase or a modified T7 RNA
polymerase, and purifying the transcribed library. The RNA or DNA
library is then mixed with the target under conditions favorable
for binding and subjected to step-wise iterations of binding,
partitioning and amplification, using the same general selection
scheme, to achieve virtually any desired criterion of binding
affinity and selectivity. More specifically, starting with a
mixture containing the starting pool of nucleic acids, the
SELEX.TM. method includes steps of: (a) contacting the mixture with
the target under conditions favorable for binding; (b) partitioning
unbound nucleic acids from those nucleic acids which have bound
specifically to target molecules; (c) optionally dissociating the
nucleic acid-target complexes; (d) amplifying the nucleic acids
dissociated from the nucleic acid-target complexes to yield a
ligand-enriched mixture of nucleic acids; and (e) reiterating the
steps of binding, partitioning, dissociating and amplifying through
as many cycles as desired to yield highly specific, high affinity
nucleic acid ligands to the target molecule. In those instances
where RNA aptamers are being selected, the SELEX.TM. method further
comprises the steps of: (i) reverse transcribing the nucleic acids
dissociated from the nucleic acid-target complexes before
amplification in step (d); and (ii) transcribing the amplified
nucleic acids from step (d) before restarting the process.
Within a nucleic acid mixture containing a large number of possible
sequences and structures, there is a wide range of binding
affinities for a given target. A nucleic acid mixture comprising,
for example, a 20 nucleotide randomized segment can have 4.sup.20
candidate possibilities. Those which have the higher affinity
(lower dissociation constants) for the target are most likely to
bind to the target. After partitioning, dissociation and
amplification, a second nucleic acid mixture is generated, enriched
for the higher binding affinity candidates. Additional rounds of
selection progressively favor the best ligands until the resulting
nucleic acid mixture is predominantly composed of only one or a few
sequences. These can then be cloned, sequenced and individually
tested as ligands or aptamers for 1) target binding affinity;
and/or 2) ability to effect target function
Cycles of selection and amplification are repeated until a desired
goal is achieved. In the most general case, selection/amplification
is continued until no significant improvement in binding strength
is achieved on repetition of the cycle. The method is typically
used to sample approximately 10.sup.14 different nucleic acid
species but may be used to sample as many as about 10.sup.18
different nucleic acid species. Generally, nucleic acid aptamer
molecules are selected in a 5 to 20 cycle procedure. In one
embodiment, heterogeneity is introduced only in the initial
selection stages and does not occur throughout the replicating
process.
In one embodiment of the SELEX.TM. method, the selection process is
so efficient at isolating those nucleic acid ligands that bind most
strongly to the selected target, that only one cycle of selection
and amplification is required. Such an efficient selection may
occur, for example, in a chromatographic-type process wherein the
ability of nucleic acids to associate with targets bound on a
column operates in such a manner that the column is sufficiently
able to allow separation and isolation of the highest affinity
nucleic acid ligands.
In many cases, it is not necessarily desirable to perform the
iterative steps of the SELEX.TM. process until a single nucleic
acid ligand is identified. The target-specific nucleic acid ligand
solution may include a family of nucleic acid structures or motifs
that have a number of conserved sequences and a number of sequences
which can be substituted or added without significantly affecting
the affinity of the nucleic acid ligands to the target. By
terminating the SELEX.TM. process prior to completion, it is
possible to determine the sequence of a number of members of the
nucleic acid ligand solution family.
A variety of nucleic acid primary, secondary and tertiary
structures are known to exist. The structures or motifs that have
been shown most commonly to be involved in non-Watson-Crick type
interactions are referred to as hairpin loops, symmetric and
asymmetric bulges, pseudoknots and myriad combinations of the same.
Almost all known cases of such motifs suggest that they can be
formed in a nucleic acid sequence of no more than 30 nucleotides.
For this reason, it is often preferred that SELEX.TM. procedures
with contiguous randomized segments be initiated with nucleic acid
sequences containing a randomized segment of between about 20 to
about 50 nucleotides and in some embodiments, about 30 to about 40
nucleotides. In one example, the 5'-fixed:random:3'-fixed sequence
comprises a random sequence of about 30 to about 40
nucleotides.
The core SELEX.TM. method has been modified to achieve a number of
specific objectives. For example, U.S. Pat. No. 5,707,796 describes
the use of the SELEX.TM. process in conjunction with gel
electrophoresis to select nucleic acid molecules with specific
structural characteristics, such as bent DNA. U.S. Pat. No.
5,763,177 describes SELEX.TM. based methods for selecting nucleic
acid ligands containing photo reactive groups capable of binding
and/or photo-crosslinking to and/or photo-inactivating a target
molecule. U.S. Pat. No. 5,567,588 and U.S. Pat. No. 5,861,254
describe SELEX.TM. based methods which achieve highly efficient
partitioning between oligonucleotides having high and low affinity
for a target molecule. U.S. Pat. No. 5,496,938 describes methods
for obtaining improved nucleic acid ligands after the SELEX.TM.
process has been performed. U.S. Pat. No. 5,705,337 describes
methods for covalently linking a ligand to its target.
The SELEX.TM. method can also be used to obtain nucleic acid
ligands that bind to more than one site on the target molecule, and
to obtain nucleic acid ligands that include non-nucleic acid
species that bind to specific sites on the target. The SELEX.TM.
method provides means for isolating and identifying nucleic acid
ligands which bind to any envisionable target, including large and
small biomolecules such as nucleic acid-binding proteins and
proteins not known to bind nucleic acids as part of their
biological function as well as cofactors and other small molecules.
For example, U.S. Pat. No. 5,580,737 discloses nucleic acid
sequences identified through the SELEX.TM. method which are capable
of binding with high affinity to caffeine and the closely related
analog, theophylline.
The Counter-SELEX.TM. process is a method for improving the
specificity of nucleic acid ligands to a target molecule by
eliminating nucleic acid ligand sequences with cross-reactivity to
one or more non-target molecules. The Counter-SELEX.TM. process is
comprised of the steps of: (a) preparing a candidate mixture of
nucleic acids; (b) contacting the candidate mixture with the
target, wherein nucleic acids having an increased affinity to the
target relative to the candidate mixture may be partitioned from
the remainder of the candidate mixture; (c) partitioning the
increased affinity nucleic acids from the remainder of the
candidate mixture; (d) optionally dissociating the increased
affinity nucleic acids from the target; (e) contacting the
increased affinity nucleic acids with one or more non-target
molecules such that nucleic acid ligands with specific affinity for
the non-target molecule(s) are removed; and (f) amplifying the
nucleic acids with specific affinity only to the target molecule to
yield a mixture of nucleic acids enriched for nucleic acid
sequences with a relatively higher affinity and specificity for
binding to the target molecule. As described above for the
SELEX.TM. method, cycles of selection and amplification are
repeated as necessary until a desired goal is achieved.
One potential problem encountered in the use of nucleic acids as
therapeutics and vaccines is that oligonucleotides in their
phosphodiester form may be quickly degraded in body fluids by
intracellular and extracellular enzymes such as endonucleases and
exonucleases before the desired effect is manifest. The SELEX.TM.
method thus encompasses the identification of high-affinity nucleic
acid ligands containing modified nucleotides conferring improved
characteristics on the ligand, such as improved in vivo stability
or improved delivery characteristics. Examples of such
modifications include chemical substitutions at the sugar and/or
phosphate and/or base positions. SELEX.TM.-identified nucleic acid
ligands containing modified nucleotides are described, e.g., in
U.S. Pat. No. 5,660,985, which describes oligonucleotides
containing nucleotide derivatives chemically modified at the
2'-position of ribose, 5-position of pyrimidines, and 8-position of
purines, U.S. Pat. No. 5,756,703 which describes oligonucleotides
containing various 2'-modified pyrimidines, and U.S. Pat. No.
5,580,737 which describes highly specific nucleic acid ligands
containing one or more nucleotides modified with 2'-amino
(2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe)
substituents.
Modifications of the nucleic acid ligands contemplated in this
invention include, but are not limited to, those which provide
other chemical groups that incorporate additional charge,
polarizability, hydrophobicity, hydrogen bonding, electrostatic
interaction, and fluxionality to the nucleic acid ligand bases or
to the nucleic acid ligand as a whole. Modifications to generate
oligonucleotide populations which are resistant to nucleases can
also include one or more substitute internucleotide linkages,
altered sugars, altered bases, or combinations thereof. Such
modifications include, but are not limited to, 2'-position sugar
modifications, 5-position pyrimidine modifications, 8-position
purine modifications, modifications at exocyclic amines,
substitution of 4-thiouridine, substitution of 5-bromo or
5-iodo-uracil; backbone modifications, phosphorothioate or alkyl
phosphate modifications, methylations, and unusual base-pairing
combinations such as the isobases isocytidine and isoguanosine.
Modifications can also include 3' and 5' modifications such as
capping. Modifications can also include 3' and 5' modifications
such as capping, e.g., addition of a 3'-3'-dT cap to increase
exonuclease resistance (see, e.g., U.S. Pat. Nos. 5,674,685;
5,668,264; 6,207,816; and 6,229,002, each of which is incorporated
by reference herein in its entirety).
In one embodiment, oligonucleotides are provided in which the P(O)O
group is replaced by P(O)S ("thioate"), P(S)S ("dithioate"),
P(O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH.sub.2
("formacetal") or 3'-amine (--NH--CH.sub.2--CH.sub.2--), wherein
each R or R' is independently H or substituted or unsubstituted
alkyl. Linkage groups can be attached to adjacent nucleotides
through an --O--, --N--, or --S-- linkage. Not all linkages in the
oligonucleotide are required to be identical.
In further embodiments, the oligonucleotides comprise modified
sugar groups, for example, one or more of the hydroxyl groups is
replaced with halogen, aliphatic groups, or functionalized as
ethers or amines. In one embodiment, the 2'-position of the
furanose residue is substituted by any of an O-methyl, O-alkyl,
O-allyl, S-alkyl, S-allyl, or halo group. Methods of synthesis of
2'-modified sugars are described, e.g., in Sproat, et al., Nucl.
Acid Res. 19:733-738 (1991); Cotten, et al., Nucl. Acid Res.
19:2629-2635 (1991); and Hobbs, et al., Biochemistry 12:5138-5145
(1973). Other modifications are known to one of ordinary skill in
the art. Such modifications may be pre-SELEX.TM. process
modifications or post-SELEX.TM. process modifications (modification
of previously identified unmodified ligands) or may be made by
incorporation into the SELEX.TM. process.
Pre-SELEX.TM. process modifications or those made by incorporation
into the SELEX.TM. process yield nucleic acid ligands with both
specificity for their SELEX.TM. target and improved stability,
e.g., in vivo stability. Post-SELEX.TM. process modifications
((e.g., truncation, deletion, substitution or additional nucleotide
modifications of previously identified ligands having nucleotides
incorporated by pre-SELEX.TM. process modification) to nucleic acid
ligands can result in improved stability, e.g., in vivo stability
without adversely affecting the binding capacity of the nucleic
acid ligand. Optionally, aptamers in which modified nucleotides
have been incorporated by pre-SELEX.TM. process modification can be
further modified by post-SELEX.TM. process modification (i.e., a
post-SELEX.TM. modification process after SELEX).
The SELEX.TM. method encompasses combining selected
oligonucleotides with other selected oligonucleotides and
non-oligonucleotide functional units as described in U.S. Pat. No.
5,637,459 and U.S. Pat. No. 5,683,867. The SELEX.TM. method further
encompasses combining selected nucleic acid ligands with lipophilic
or non-immunogenic high molecular weight compounds in a diagnostic
or therapeutic complex, as described, e.g., in U.S. Pat. No.
6,011,020, U.S. Pat. No. 6,051,698, and PCT Publication No. WO
98/18480. These patents and applications teach the combination of a
broad array of shapes and other properties, with the efficient
amplification and replication properties of oligonucleotides, and
with the desirable properties of other molecules.
The identification of nucleic acid ligands to small, flexible
peptides via the SELEX.TM. method has also been explored. Small
peptides have flexible structures and usually exist in solution in
an equilibrium of multiple conformers, and thus it was initially
thought that binding affinities may be limited by the
conformational entropy lost upon binding a flexible peptide.
However, the feasibility of identifying nucleic acid ligands to
small peptides in solution was demonstrated in U.S. Pat. No.
5,648,214. In this patent, high affinity RNA nucleic acid ligands
to substance P, an 11 amino acid peptide, were identified.
As part of the SELEX.TM. process, the sequences selected to bind to
the target are then optionally minimized to determine the minimal
sequence having the desired binding affinity. The selected
sequences and/or the minimized sequences are optionally modified by
performing random or directed mutagenesis of the sequence to, e.g.,
increase binding affinity or alternatively to determine which
positions in the sequence are essential for binding activity.
The 2'-Modified SELEX.TM. Method
In order for an aptamer to be suitable for use as a therapeutic
and/or for particular types of diagnostics, it is preferably
inexpensive to synthesize, safe and stable in vivo. Wild-type RNA
and DNA aptamers are typically not stable in vivo because of their
susceptibility to degradation by nucleases. Resistance to nuclease
degradation can be greatly increased by the incorporation of
modifying groups at the 2'-position.
2'-fluoro and 2'-amino groups have been successfully incorporated
into oligonucleotide pools from which aptamers have been
subsequently selected. However, these modifications greatly
increase the cost of synthesis of the resultant aptamer, and may
introduce safety concerns in some cases because of the possibility
that the modified nucleotides could be recycled into host DNA by
degradation of the modified oligonucleotides and subsequent use of
the nucleotides as substrates for DNA synthesis.
Aptamers that contain 2'-O-methyl ("2'-OMe") nucleotides, as
provided herein, overcome many of these drawbacks. Oligonucleotides
containing 2'-OMe nucleotides are nuclease-resistant and
inexpensive to synthesize. Although 2'-OMe nucleotides are
ubiquitous in biological systems, natural polymerases do not accept
2'-OMe NTPs as substrates under physiological conditions, thus
there are no safety concerns over the recycling of 2'-OMe
nucleotides into host DNA. SELEX.TM. methods used to generate
2'-modified aptamers are described, e.g., in U.S. Provisional
Patent Application Ser. No. 60/430,761, filed Dec. 3, 2002, U.S.
Provisional Patent Application Ser. No. 60/487,474, filed Jul. 15,
2003, U.S. Provisional Patent Application Ser. No. 60/517,039,
filed Nov. 4, 2003, U.S. patent application Ser. No. 10/729,581,
filed Dec. 3, 2003, and U.S. patent application Ser. No.
10/873,856, filed Jun. 21, 2004, entitled "Method for in vitro
Selection of 2'-O-methyl Substituted Nucleic Acids," each of which
is herein incorporated by reference in its entirety.
The present invention includes aptamers that bind to and modulate
the function of the aptamer target and which contain modified
nucleotides (e.g., nucleotides which have a modification at the
2'-position) to make the oligonucleotide more stable than the
unmodified oligonucleotide to enzymatic and chemical degradation as
well as thermal and physical degradation. Although there are
several examples of 2'-OMe containing aptamers in the literature
(see, e.g., Ruckman et al., J.Biol.Chem, 1998 273, 20556-20567-695)
these were generated by the in vitro selection of libraries of
modified transcripts in which the C and U residues were 2'-fluoro
(2'-F) substituted and the A and G residues were 2'-OH. Once
functional sequences were identified then each A and G residue was
tested for tolerance to 2'-OMe substitution, and the aptamer was
re-synthesized having all A and G residues which tolerated 2'-OMe
substitution as 2'-OMe residues. Most of the A and G residues of
aptamers generated in this two-step fashion tolerate substitution
with 2'-OMe residues, although, on average, approximately 20% do
not. Consequently, aptamers generated using this method tend to
contain from two to four 2'-OH residues, and stability and cost of
synthesis are compromised as a result. By incorporating modified
nucleotides into the transcription reaction which generate
stabilized oligonucleotides used in oligonucleotide pools from
which aptamers are selected and enriched by the SELEX.TM. method
(and/or any of its variations and improvements, including those
described herein), the methods of the present invention eliminate
the need for stabilizing the selected aptamer oligonucleotides by
resynthesizing the aptamer oligonucleotides with 2'-OMe modified
nucleotides.
In one embodiment, the present invention provides aptamers
comprising combinations of 2'-OH, 2'-F, 2'-deoxy, and 2'-OMe
modifications of the ATP, GTP, CTP, TTP, and UTP nucleotides. In
another embodiment, the present invention provides aptamers
comprising combinations of 2'-OH, 2'-F, 2'-deoxy, 2'-OMe,
2'-NH.sub.2, and 2'-methoxyethyl modifications of the ATP, GTP,
CTP, TTP, and UTP nucleotides. In a preferred embodiment, the
present invention provides aptamers comprising all or substantially
all 2'-OMe modified ATP, GTP, CTP, TTP, and/or UTP nucleotides.
Modified Polymerases
2'-modified aptamers of the invention are created using modified
polymerases, e.g., a modified T7 polymerase, having a rate of
incorporation of modified nucleotides having bulky substituents at
the furanose 2' position that is higher than that of wild-type
polymerases. For example, a mutant T7 polymerase in which the
tyrosine residue at position 639 has been changed to phenylalanine
(Y639F) readily utilizes 2'deoxy, 2'amino-, and 2'fluoro-nucleotide
triphosphates (NTPs) as substrates and has been widely used to
synthesize modified RNAs for a variety of applications. However,
this mutant T7 polymerase reportedly can not readily utilize (i.e.,
incorporate) NTPs with bulky 2'-substituents such as 2'-OMe or
2'-azido (2'-N.sub.3) substituents. For incorporation of bulky 2'
substituents, a mutant T7 polymerase having the histidine at
position 784 changed to an alanine residue in addition to the Y639F
mutation has been described (Y639F/H784A) and has been used in
limited circumstances to incorporate modified pyrimidine NTPs. See
Padilla, R. and Sousa, R., Nucleic Acids Res., 2002, 30(24): 138. A
mutant T7 RNA polymerase in which the tyrosine residue at position
639 has been changed to phenylalanine, the histidine residue at
position 784 has been changed to an alanine, and the lysine residue
at position 378 has been changed to arginine (Y639F/H784A/K378R)
has been used in limited circumstances to incorporate modified
purine and pyrimidine NTPs, e.g., 2'-OMe NTPs, but includes a spike
of 2'-OH GTP for transcription. See Burmeister et. al., (2005)
Chemistry and Biology, 12: 25-33. The inclusion of a 2'-OH GTP
spike for transcription may result in aptamers that are not fully
2'-OMe but rather may depend on the presence of 2'-OH GTPs.
A mutant T7 polymerase having the histidine at position 784 changed
to an alanine residue (H784A) has also been described. Padilla et
al., Nucleic Acids Research, 2002, 30: 138. In both the Y639F/H784A
mutant and H784A mutant T7 polymerases, the change to a smaller
amino acid residue such as alanine allows for the incorporation of
bulkier nucleotide substrates, e.g., 2'-OMe substituted
nucleotides. See Chelliserry, K. and Ellington, A. D., (2004)
Nature Biotech, 9:1155-60. Additional T7 RNA polymerases have been
described with mutations in the active site of the T7 RNA
polymerase which more readily incorporate bulky 2'-modified
substrates, e.g., a mutant T7 RNA polymerase having the tyrosine
residue at position 639 changed to a leucine (Y639L). However
activity is often sacrificed for increased substrate specificity
conferred by such mutations, leading to low transcript yields. See
Padilla R and Sousa, R., (1999) Nucleic Acids Res., 27(6): 1561.
The T7 RNA polymerase mutant P266L has been described to facilitate
promoter clearance (Guillerez et al. (2005) Proc. Nat. Acad. Sci.
USA, 102 (17) 5958). The polymerase makes a transition from the
initiation conformation, in which it is bound to the promoter, to
the elongation conformation in which it is not. None of the above
mutant polymerases were reported to result in fully 2'-OMe
transcripts.
The present invention provides materials and methods for increasing
the transcription yield of oligonucleotides. In one embodiment, the
present invention provides methods and conditions for using
modified T7 RNA polymerases to enzymatically incorporate modified
nucleotides into oligonucleotides. In a preferred embodiment, the
modified T7 RNA polymerase used with the transcription methods of
the invention does not require the presence of 2'-OH GTP. In a
preferred embodiment, the modified polymerase is a mutant T7 RNA
polymerase having the tyrosine residue at position 639 changed to a
leucine residue and the histidine residue at position 784 changed
to an alanine residue (Y639L/H784A). In another preferred
embodiment, the modified polymerase is a mutant T7 RNA polymerase
having the tyrosine residue at position 639 changed to a leucine
residue, the histidine residue at position 784 changed to an
alanine residue, and the lysine residue at position 378 changed to
an arginine residue (Y639L/H784A/K378R). In another embodiment, the
modified polymerase for use in the methods of the invention is a
mutant T7 RNA polymerase having the tyrosine residue at position
639 changed to a leucine (Y639L) while in yet another embodiment
the mutant T7 RNA polymerase has the tyrosine residue at position
639 changed to a leucine residue and the lysine residue at position
378 changed to an arginine residue (Y639L/K378R). While not wishing
to be bound by any theory, the K378R mutation is not near the
active site of the polymerase and thus is believed to be a silent
mutation. In another embodiment, the modified polymerase for use in
the methods of the invention is a mutant T7 RNA polymerase having
the proline residue at position 266 changed to a leucine, the
tyrosine residue at position 639 changed to a leucine and the
histidine residue at position 784 changed to an alanine residue,
(P266L/Y639L/H784A) while in yet another embodiment the mutant T7
RNA polymerase has the proline residue at position 266 changed to a
leucine, the tyrosine residue at position 639 changed to a leucine
residue, the histidine residue at position 784 changed to an
alanine residue and the lysine residue at position 378 changed to
an arginine residue (P266L/Y639L/H784A/K378R).
The amino acid sequences of the mutant T7 RNA polymerases are shown
below:
TABLE-US-00001 Y639L/H784A (SEQ ID NO 1):
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLALGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLK
SAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLM
FLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVASQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFA
DQLHESQLDKMPALPAKGNLNLRDILESDFAFA Y639L/H784A/K378R (SEQ ID NO 2):
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWRRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLALGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLK
SAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLM
FLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVASQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFA
DQLHESQLDKMPALPAKGNLNLRDILESDFAFA Y639L (SEQ ID NO 100):
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLALGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLK
SAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLM
FLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFA
DQLHESQLDKMPALPAKGNLNLRDILESDFAFA Y639L/K378R (SEQ ID NO 101):
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWRRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLALGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTWAAVEAMNWLKS
AAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMF
LGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEK
YGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFAD
QLHESQLDKMPALPAKGNLNLRDILESDFAFA P266L/Y639L/H784A (SEQ ID NO 102)
MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLALGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLK
SAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLM
FLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVASQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFA
DQLHESQLDKMPALPAKGNLNLRDILESDFAFA P266L/Y639L/H784A/K378R (SEQ ID
NO 103) MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEAR
FRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRP
TAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEAR
FGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEA
WSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEY
AEAIATRAGALAGISLMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTH
SKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVE
DIPAIEREELPMKPEDIDMNPEALTAWRRAAAAVYRKDKARKSRRISLEF
MLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGK
PIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENT
WWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAML
RDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDE
NTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLALGSKEFGFRQQV
LEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLK
SAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLM
FLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVASQDGSHLRKTVVWAHE
KYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFA
DQLHESQLDKMPALPAKGNLNLRDILESDFAFA
To generate pools of 2'-modified (e.g., 2'-OMe) RNA transcripts
under conditions in which a polymerase accepts 2'-modified NTPs,
the Y639F, Y639F/K378R, Y639F/H784A, Y639F/H784A/K378R,
Y639L/H784A, Y639L/H784A/K378R, Y639L, Y639L/K378R,
P266L/Y639L/H784A or P266L/Y639L/H784A/K378R mutant T7 RNA
polymerases can be used. A preferred polymerase is the Y639L/H784A
mutant T7 RNA polymerase. Another preferred polymerase is the
Y639L/H784A/K378R mutant T7 RNA polymerase. Another preferred
polymerase of the invention is the P266L/Y639L/H784A or
P266L/Y639L/H784A/K378R mutant T7 RNA polymerase. Other T7 RNA
polymerases, particularly those that exhibit a high tolerance for
bulky 2'-substituents, may also be used in the methods of the
present invention. When used in a template-directed polymerization
using the conditions disclosed herein, the Y639L/H784A, the
Y639L/H784A/K378R, P266L/Y639L/H784A or P266L/Y639L/H784A/K378R
mutant T7 RNA polymerase can be used for the incorporation of all
2'-OMe NTPs, including 2'-OMe GTP, with higher transcript yields
than achieved by using the Y639F, Y639F/K378R, Y639F/H784A,
Y639F/H784A/K378R, Y639L, or the Y639L/K378R mutant T7 RNA
polymerases. The Y639L/H784A, Y639L/H784A/K378R, P266L/Y639L/H784A
or P266L/Y639L/H784A/K378R mutant T7 RNA polymerases can be used
with but does not require 2'-OH GTP to achieve high yields of
2'-modified, e.g., 2'-OMe containing oligonucleotides.
In a preferred embodiment, the Y639L/H784A or the Y639L/H784A/K378R
mutant T7 RNA polymerases of the invention are used with an MNA
transcription mixture to promote higher fully 2'-OMe transcript
yields. In some embodiments, Y639L/H784A or the Y639L/H784A/K378R
mutant T7 RNA polymerases may be used with an rRmY, dRmY, rGmH,
fGmH, dGmH, dAmB, rRdY, dRdY or rN transcription mixture.
As used herein, a transcription mixture containing only 2'-OMe A,
G, C, and U triphosphates is referred to as an MNA mixture, and
aptamers selected therefrom are referred to as MNA aptamers and
contains only 2'-O-methyl nucleotides. A transcription mixture
containing 2'-OMe C and U and 2'-OH A and G is referred to as an
"rRmY" mixture and aptamers selected therefrom are referred to as
"rRmY" aptamers. A transcription mixture containing deoxy A and G
and 2'-OMe U and C is referred to as a "dRmY" mixture and aptamers
selected therefrom are referred to as "dRmY" aptamers. A
transcription mixture containing 2'-OMe A, C, and U, and 2'-OH G is
referred to as a "rGmH" mixture and aptamers selected therefrom are
referred to as "rGmH" aptamers. A transcription mixture alternately
containing 2'-OMe A, C, U and G and 2'-OMe A, U and C and 2'-F G is
referred to as an "alternating mixture" and aptamers selected
therefrom are referred to as "alternating mixture" aptamers. A
transcription mixture containing 2'-OMe A, U, and C, and 2'-F G is
referred to as a "fGmH" mixture and aptamers selected therefrom are
referred to as "fGmH" aptamers. A transcription mixture containing
2'-OMe A, U, and C, and deoxy G is referred to as a "dGmH" mixture
and aptamers selected therefrom are referred to as "dGmH" aptamers.
A transcription mixture containing deoxy A, and 2'-OMe C, G and U
is referred to as a "dAmB" mixture and aptamers selected therefrom
are referred to as "dAmB" aptamers. A transcription mixture
containing 2'-OH A and 2'-OMe C, G and U is referred to as a "rAmB"
mixture and aptamers selected therefrom are referred to as "rAmB"
aptamers. A transcription mixture containing 2'-OH adenosine
triphosphate and guanosine triphosphate and deoxy cytidine
triphosphate and thymidine triphosphate is referred to as an rRdY
mixture and aptamers selected therefrom are referred to as "rRdY"
aptamers. A transcription mixture containing all 2'-OH nucleotides
is referred to as a "rN" mixture and aptamers selected therefrom
are referred to as "rN", "rRrY" or RNA aptamers, and a
transcription mixture containing all deoxy nucleotides is referred
to as a "dN" mixture and aptamers selected therefrom are referred
to as "dN" or "dRdY" or DNA aptamers.
2'-modified oligonucleotides may be synthesized entirely of
modified nucleotides, or with a subset of modified nucleotides. All
nucleotides may be modified, and all may contain the same
modification. All nucleotides may be modified, but contain
different modifications, e.g., all nucleotides containing the same
base may have one type of modification, while nucleotides
containing other bases may have different types of modification.
All purine nucleotides may have one type of modification (or are
unmodified), while all pyrimidine nucleotides have another,
different type of modification (or are unmodified). In this manner,
transcripts, or pools of transcripts are generated using any
combination of modifications, including for example,
ribonucleotides (2'-OH), deoxyribonucleotides (2'-deoxy), 2'-F, and
2'-OMe nucleotides. Additionally modified oligonucleotides may
contain nucleotides bearing more than one modification
simultaneously such as a modification at the internucleotide
linkage (eg phosphorothioate) and at the sugar (eg 2'-OMe) and the
base (eg inosine).
Transcription Conditions
A number of factors have been determined to be important for the
transcription conditions of the 2'-modified SELEX.TM. method, which
may also apply to the Terminal Region SELEX.TM. methods described
below. For example, increases in the yields of modified transcript
may be observed under some conditions when a particular leader
sequence/mutant polymerase combination is used. A leader sequence
is a sequence that can be incorporated into the 3' end of a fixed
sequence at the 5' end of the DNA transcription template. The
leader sequence is typically 6-15 nucleotides long, and may be
composed of a predetermined nucleotide composition, for example it
may be all purines, or a particular mixture of purine and
pyrimidine nucleotides.
Examples of templates that may be used with the mutant polymerases
and transcription conditions of the invention, particularly in
combination with Y639L/H784A, Y639L/H784A/K378R, P266L/Y639L/H784A
or P266L/Y639L/H784A/K378R, are ARC2118 (SEQ ID NO 3), ARC2119 (SEQ
ID NO 4), and ARC3428
GGGAGACAAGAATAAAGCGAGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAAGAGTCGATGA
TGCTTAGCTAG (SEQ ID NO 137).
In addition, the presence of 2'-OH GTP has historically been an
important factor in obtaining transcripts incorporating modified
nucleotides. Transcription can be divided into two phases: the
first phase is initiation, during which an NTP is added to the
3'-end of GTP (or another substituted guanosine) to yield a
dinucleotide which is then extended by about 10-12 nucleotides; the
second phase is elongation, during which transcription proceeds
beyond the addition of the first about 10-12 nucleotides. It was
previously found that small amounts of 2'-OH GTP added to a
transcription mixture containing Y639F/K378R mutant or
Y639F/H784A/K378R mutant T7 RNA polymerase and an excess of 2'-OMe
GTP was sufficient to enable the polymerase to initiate
transcription using 2'-OH GTP (and gave a higher yield of 2'-OMe
containing transcript than without 2'-OH GTP), but once
transcription enters the elongation phase the reduced
discrimination between 2'-OMe and 2'-OH GTP, and the excess of
2'-OMe GTP over 2'-OH GTP allows the incorporation of principally
the 2'-OMe GTP.
The present invention provides mutant T7 RNA polymerases, e.g
Y639L/H784A, Y639L/H784A/K378R, P266L/Y639L/H784A or
P266L/Y639L/H784A/K378R which do not require 2'-OH GTP in the
transcription mixture for a high yield of 2'-OMe transcription. In
one embodiment, high yield means on average at least one transcript
per input transcription template.
Another factor in the incorporation of 2'-OMe substituted
nucleotides into transcripts is the use of both divalent magnesium
and manganese (Mn.sup.2+) in the transcription mixture. Different
combinations of concentrations of magnesium chloride and manganese
chloride have been found to affect yields of 2'-O-methylated
transcripts, the optimum concentration of the magnesium and
manganese chloride being dependent on the concentration in the
transcription reaction mixture of NTPs which complex divalent metal
ions. To obtain the greatest yields of all 2'-O-methylated
transcripts (i.e., all 2'-OMe A, C, U and G nucleotides),
concentrations of approximately 5 mM magnesium chloride and 1.5 mM
manganese chloride are preferred when each NTP is present at a
concentration of 0.5 mM. When the concentration of each NTP is 1.0
mM, concentrations of approximately 6.5 mM magnesium chloride and
2.0 mM manganese chloride are preferred. When each NTP is present
at a concentration of 1.5 mM, concentrations of approximately 8 mM
magnesium chloride and 2.5 mM manganese chloride are preferred.
When the concentration of each NTP is 2.0 mM, concentrations of
approximately 9.5 mM magnesium chloride and 3.0 mM manganese
chloride are preferred. In any case, departures from these
concentrations of up to two-fold still give significant amounts of
modified transcripts.
Priming transcription with 2'-OH GMP, guanosine, or other 2'-OH
guanosines substituted at a position other than the 2'-OH sugar
position is also important for transcription mixtures which do not
contain 2'-OH GTP. This effect results from the specificity of the
polymerase for the initiating nucleotide. As a result, the
5'-terminal nucleotide of any transcript generated in this fashion
is likely to be 2'-OH G. A preferred concentration of GMP (or
guanosine) is 0.5 mM and even more preferably 1 mM. It has also
been found that including PEG, preferably PEG-8000, in the
transcription reaction is useful to maximize incorporation of
modified nucleotides.
For maximum incorporation of 2'-OMe ATP (100%), 2'-OMe UTP (100%),
2'-OMe CTP (100%) and 2'-OMe GTP (100%) ("MNA") into transcripts
the following conditions may be used: HEPES buffer 200 mM, DTT 40
mM, spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v),
MgCl.sub.2 8 mM, MnCl.sub.2 2.5 mM, 2'-OMe NTP (each) 1.5 mM, 2'-OH
GMP 1 mM, pH 7.5, Y639L/H784A/K378R mutant T7 RNA Polymerase 200
nM, inorganic pyrophosphatase 5 units/ml, and a DNA template. In
some embodiments, the DNA template may be present in a
concentration of preferably about 5 to 500 nM. Optionally, the DNA
template used with the above transcription conditions comprises an
all purine leader sequence that increases the transcription yield
relative to a template that does not comprise such a leader
sequence when both templates are transcribed under identical
conditions. In another embodiment, the leader sequence is a mixture
of purines and pyrimidines that increases the transcription yield
relative to a template that does not comprise such a leader
sequence when both are transcribed under identical conditions. As
used herein, one unit of inorganic pyrophosphatase is defined as
the amount of enzyme that will liberate 1.0 mole of inorganic
orthophosphate per minute at pH 7.2 and 25.degree. C. The reaction
may be carried out from about 1 to 24 hours.
In each case, the transcription products can then be used for input
into the SELEX.TM. process to identify aptamers and/or to determine
a conserved sequence that has binding specificity to a given
target. The resulting sequences are already stabilized, eliminating
this step from the post-SELEX.TM. modification process and giving a
more highly stabilized aptamer as a result.
As described below, useful yields of transcripts fully
incorporating 2' substituted nucleotides can be obtained under
conditions other than the conditions described above. For example,
variations to the above transcription conditions include:
The HEPES buffer concentration can range from 0 to 1 M. The present
invention also contemplates the use of other buffering agents
having a pKa between 5 and 10 including, for example,
Tris-hydroxymethyl-aminomethane.
The DTT concentration can range from 0 to 400 mM. The methods of
the present invention also provide for the use of other reducing
agents including, for example, mercaptoethanol.
The spermidine and/or spermine concentration can range from 0 to 20
mM.
The PEG-8000 concentration can range from 0 to 50% (w/v). The
methods of the present invention also provide for the use of other
hydrophilic polymer including, for example, other molecular weight
PEG or other polyalkylene glycols.
The Triton X-100 concentration can range from 0 to 0.1% (w/v). The
methods of the present invention also provide for the use of other
non-ionic detergents including, for example, other detergents,
including other Triton-X detergents.
The MgCl.sub.2 concentration can range from 0.5 mM to 50 mM. The
MnCl.sub.2 concentration can range from 0.15 mM to 15 mM. Both
MgCl.sub.2 and MnCl.sub.2 must be present within the ranges
described and in a preferred embodiment are present in about a 10
to about 3 ratio of MgCl.sub.2:MnCl.sub.2, preferably, the ratio is
about 3-5:1, more preferably, the ratio is about 3-4:1.
The 2'-OMe NTP concentration (each NTP) can range from 5 .mu.M to 5
mM.
The 2'-OH GTP concentration can range from 0 .mu.M to 300 .mu.M. In
a preferred embodiment, transcription occurs in the absence of
2'-OH GTP (0 .mu.M).
The concentration of 2'-OH GMP, guanosine or other 2'-OH G
substituted at a position other than the 2'sugar position, can
range from 0 to 5 mM. Where 2'-OH GTP is not included in the
reaction 2'-OH GMP is required and may range from 5 nM to 5
.mu.M.
The DNA template concentration can range from 5 nM to 5 .mu.M.
The mutant polymerase concentration can range from 2 nM to 20
.mu.M.
The inorganic pyrophosphatase can range from 0 to 100 units/ml.
The pH can range from pH 6 to pH 9. The methods of the present
invention can be practiced within the pH range of activity of most
polymerases that incorporate modified nucleotides.
The transcription reaction may be allowed to occur from about one
hour to weeks, preferably from about 1 to about 24 hours.
In addition, the methods of the present invention provide for the
optional use of chelating agents in the transcription reaction
condition including, for example, EDTA, EGTA, and DTT.
Terminal Region SELEX.TM. Method
A method for the discovery of nucleic acid transcription template
sequences that in some embodiments are used to program a
template-directed nucleotide triphosphate polymerization will
increase the transcript yield, is a variant of the SELEX.TM. method
known as the Terminal Region SELEX.TM. method (TR-SELEX.TM.
method). The present invention provides a method for identifying
nucleic acid transcription template component sequences, e.g.
leader sequences, the use of which increases transcript yield,
particularly the yield of transcripts containing 2'-modified
nucleotides (e.g., 2'-OMe nucleotides), when used to program a
template-directed polymerization, using the TR-SELEX.TM.
method.
To select for leader sequences which promote an increased yield of
transcripts containing 2'-modified nucleotides, a candidate library
of oligonucleotide transcription templates is generated which
contains a promoter sequence which allows for transcription in a
template dependent manner, a first fixed region comprising greater
than one fixed nucleotide immediately 3' to the promoter to allow
for splinted ligations to occur, thereby permitting amplification
by the extension of primers bound to primer binding sites on the
ligated template; a degenerate region from which the leader
sequence will be selected; and a fixed sequence at the 3' terminus
to allow for amplification. In a preferred embodiment, the
degenerate region of the library template is close to the
5'-terminus thereby reducing the length of the 5' fixed
sequence.
This library of transcription templates is optionally PCR
amplified, and then used to program transcription using a
transcription reaction mixture comprising a polymerase, (including
without limitation, a mutated T7 RNA polymerase), nucleotide
triphosphates (NTPs) (including without limitation one or more
2'-modified NTPs), and magnesium ions, under conditions disclosed
herein. The resulting transcript mixture is reverse transcribed to
obtain a candidate mixture of cDNA sequences which are then ligated
to a DNA sequence encoding the T7 promoter. Optionally, the
resulting transcript mixture first undergoes ligation, and is then
reverse transcribed. The cDNA which encodes the transcripts are
then amplified by PCR, and clones are assayed for transcription
yield using gel analysis. Transcription templates amplified in this
manner can optionally be used to perform further rounds of the
TR-SELEX.TM. process if necessary to achieve greater transcript
yield (See FIG. 2).
Clone sequences of the amplified transcripts can be analyzed to
identify the 5'-leader sequence element which allows for
transcription (including without limitation transcription
incorporating one or more 2'-modified nucleotides). These 5' leader
sequence elements are useful for designing candidate libraries of
oligonucleotide transcription templates which may be used in
SELEX.TM. to promote an increased yield of nucleic acid transcripts
which contain 2'-modified nucleotides. Examples of preferred
libraries of DNA transcription templates which incorporate
5'-leader sequence elements identified by the TR-SELEX.TM. method
(shown underlined) and promote higher yields of transcripts
containing 2'-modified nucleotides, e.g., 2'-OMe nucleotides, using
the conditions disclosed herein are described below.
For each of the sequences of the libraries of DNA transcription
templates listed below, the 5'-leader sequence element is shown
underlined, and all sequences are in the 5'-3' direction.
TABLE-US-00002 ARC 2118 (SEQ ID NO 3)
TAATACGACTCACTATAGGGGAGTACAATAACGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2119 (SEQ ID NO 4)
TAATACGACTCACTATAGGGGGTGATATTGACGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2120 (SEQ ID NO 5)
TAATACGACTCACTATAGGGGTGCGCGGTTACGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2121 (SEQ ID NO 6)
TAATACGACTCACTATAGGGGGAGGGGGTGCCGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG
To generate transcript mixtures of 2'-modified (e.g., 2'-OMe) RNA
transcripts under conditions in which a polymerase accepts
2'-modified NTPs, the Y639F, Y639F/K378R, Y639F/H784A,
Y639F/H784A/K378R, Y639L/H784A, Y639L/H784A/K378R,
P266L/Y639L/H784A) or P266L/Y639L/H784A/K378R) mutant T7 RNA
polymerase can be used with the 5'-leader sequences identified by
the methods provided by the present invention. A preferred
polymerase to be used with the 5' leader sequences of the present
invention, giving the highest yield of nucleic acid transcripts
containing 2'-modified nucleotides, is the Y639L/H784A mutant RNA
polymerase previously described. Another preferred polymerase to be
used with the 5'-leader sequences of the invention is the
Y639L/H784A/K378R mutant T7 RNA polymerase. Other T7 RNA
polymerases, particularly those that exhibit a high tolerance for
bulky 2'-substituents, may also be used in the present
invention.
In addition to incorporating leader sequences in candidate
libraries and mutant polymerases which promote increased yields of
nucleic acid transcripts containing 2'-modified nucleotides (e.g.,
Y639L/H784A and Y639L/H784A/K378R mutant T7 RNA polymerases), the
numerous factors described above which have been determined to be
important for the transcription conditions can be used to further
increase the yield of transcripts containing 2'-modified
nucleotides.
The identified leader sequences and the Y639F/H784A,
Y639F/H784A/K378R, Y639L, Y639L/K378R, Y639L/H874A,
Y639L/H874A/K378R, P266L/Y639L/H784A or P266L/Y639L/H784A/K378R
mutant T7 RNA polymerases, can be used in SELEX.TM. with the
conditions described herein to generate aptamers comprising any
combination of 2'-modified nucleotides, e.g., 2'-OH, 2'-F,
2'-deoxy, 2'-OMe, and 2'-NH.sub.2 modifications of the ATP, GTP,
CTP, TTP, and UTP nucleotides. The 2'-modified nucleotides
incorporated are preferably 2'-O-methyl nucleotides. An aptamer
composition comprising one or more 2'-O-methyl nucleotides is
preferred. An aptamer composition comprising 100% 2'-O-methyl
purines and pyrimidines, except for the starting nucleotide, is
more preferred. In one preferred embodiment, one of the identified
leader sequences and the Y639L/H874A, Y639L/H784A/K378R,
P266L/Y639L/H784A or P266L/Y639L/H784A/K378R mutant T7 RNA
polymerases are used in the SELEX.TM. method with the conditions
described herein to generate higher transcript yields of aptamers
comprising fully 2'-OMe nucleotides.
For maximum incorporation of 2'-OMe ATP (100%), UTP (100%), CTP
(100%) and GTP (100%) (MNA'') into transcripts the following
conditions are preferred: HEPES buffer 200 mM, DTT 40 mM,
spermidine 2 mM, PEG-8000 10% (w/v), Triton X-100 0.01% (w/v),
MgCl.sub.2 8 mM, MnCl.sub.2 2.5 mM, 2'-OMe NTP (each) 1.5 mM, 2'-OH
GMP 1 mM, pH 7.5, Y639L/H784A/K378R T7 RNA Polymerase 200 nM,
inorganic pyrophosphatase 5 units/ml, and a leader sequence that
increases the transcription yield under the derived transcription
conditions. In one embodiment, the leader sequence is an all purine
leader sequence. In another embodiment, the leader sequence is a
mixture of purines and pyrimidines. As used herein, one unit of
inorganic pyrophosphatase is defined as the amount of enzyme that
will liberate 1.0 mole of inorganic orthophosphate per minute at pH
7.2 and 25.degree. C.
Aptamer Medicinal Chemistry
Once aptamers that bind to a desired target are identified, several
techniques may be optionally performed to further increase binding
and/or functional characteristics of the identified aptamer
sequences. Aptamers, e.g. MNA aptamers, that bind to a desired
target identified through the SELEX.TM. process, (e.g. the
2'-Modified SELEX.TM. method) may be optionally truncated to obtain
the minimal aptamer sequence (also referred to herein as "minimized
construct") having the desired binding and/or functional
characteristics. One method of accomplishing this is by using
folding programs and sequence analysis (e.g., aligning clone
sequences resulting from a selection to look for conserved motifs
and/or covariation) to inform the design of minimized constructs.
Biochemical probing experiments can also be performed to determine
the 5' and 3' boundaries of an aptamer sequence to inform the
design of minimized constructs. Minimized constructs can then be
chemically synthesized and tested for binding and functional
characteristics as compared to the non-minized sequence from which
they were derived. Variants of an aptamer sequence containing a
series of 5', 3' and/or internal deletions may also be directly
chemically synthesized and tested for binding and/or functional
characteristics as compared to the non-minimized aptamer sequence
from which they were derived.
Additionally, doped reselections may be used to explore the
sequence requirements within a single active aptamer sequence such
as an MNA aptamer (i.e., an aptamer that binds to a desired target
identified through the SELEX.TM. process, (including 2'-Modified
SELEX.TM. process), or a single minimized aptamer sequence. Doped
reselections are carried out using a synthetic, degenerate pool
that has been designed based on the single sequence of interest.
The level of degeneracy usually varies 70% to 85% from the wild
type nucleotide, i.e., the single sequence of interest. In general,
sequences with neutral mutations are identified through the doped
reselection process, but in some cases sequence changes can result
in improvements in affinity. The composite sequence information
from clones identified using doped reselections can then be used to
identify the minimal binding motif and aid in Medicinal Chemistry
efforts.
Aptamer sequences identified using the SELEX.TM. process such as
MNA aptamers (including the 2'-Modified SELEX.TM. process and doped
reselections) and/or minimized aptamer sequences may also be
optionally modified post-SELEX.TM. selection using Aptamer
Medicinal Chemistry to perform random or directed mutagenesis of
the sequence to increase binding affinity and/or functional
characteristics, or alternatively to determine which positions in
the sequence are essential for binding activity and/or functional
characteristics.
Aptamer Medicinal Chemistry is an aptamer improvement technique in
which sets of variant aptamers are chemically synthesized. These
sets of variants typically differ from the parent aptamer by the
introduction of a single substituent, and differ from each other by
the location of this substituent. These variants are then compared
to each other and to the parent. Improvements in characteristics
may be profound enough that the inclusion of a single substituent
may be all that is necessary to achieve a particular therapeutic
criterion.
Alternatively the information gleaned from the set of single
variants may be used to design further sets of variants in which
more than one substituent is introduced simultaneously. In one
design strategy, all of the single substituent variants are ranked,
the top 4 are chosen and all possible double (6), triple (4) and
quadruple (1) combinations of these 4 single substituent variants
are synthesized and assayed. In a second design strategy, the best
single substituent variant is considered to be the new parent and
all possible double substituent variants that include this
highest-ranked single substituent variant are synthesized and
assayed. Other strategies may be used, and these strategies may be
applied repeatedly such that the number of substituents is
gradually increased while continuing to identify further-improved
variants.
Aptamer Medicinal Chemistry may be used particularly as a method to
explore the local, rather than the global, introduction of
substituents. Because aptamers are discovered within libraries that
are generated by transcription, any substituents that are
introduced during the SELEX.TM. process must be introduced
globally. For example, if it is desired to introduce
phosphorothioate linkages between nucleotides then they can only be
introduced at every A (or every G, C, T, U etc.) (globally
substituted). Aptamers which require phosphorothioates at some As
(or some G, C, T, U etc.) (locally substituted) but cannot tolerate
it at other As cannot be readily discovered by this process.
The kinds of substituent that can be utilized by the Aptamer
Medicinal Chemistry process are only limited by the ability to
generate them as solid-phase synthesis reagents and introduce them
into an oligomer synthesis scheme. The process is certainly not
limited to nucleotides alone. Aptamer Medicinal Chemistry schemes
may include substituents that introduce steric bulk,
hydrophobicity, hydrophilicity, lipophilicity, lipophobicity,
positive charge, negative charge, neutral charge, zwitterions,
polarizability, nuclease-resistance, conformational rigidity,
conformational flexibility, protein-binding characteristics, mass
etc. Aptamer Medicinal Chemistry schemes may include
base-modifications, sugar-modifications or phosphodiester
linkage-modifications.
When considering the kinds of substituents that are likely to be
beneficial within the context of a therapeutic aptamer, it may be
desirable to introduce substitutions that fall into one or more of
the following categories: (1) Substituents already present in the
body, e.g., 2'-deoxy, 2'-ribo, 2'-O-methyl purines or pyrimidines
or 5-methyl cytosine. (2) Substituents already part of an approved
therapeutic, e.g., phosphorothioate-linked oligonucleotides. (3)
Substituents that hydrolyze or degrade to one of the above two
categories, e.g., methylphosphonate-linked oligonucleotides. (4)
The aptamers of the present invention include aptamers developed
through aptamer medicinal chemistry as described herein.
Target binding affinity of the aptamers of the present invention
can be assessed through a series of binding reactions between the
aptamer and target (e.g., a protein) in which trace
.sup.32P-labeled aptamer is incubated with a dilution series of the
target in a buffered medium then analyzed by nitrocellulose
filtration using a vacuum filtration manifold. Referred to herein
as the dot blot binding assay, this method uses a three layer
filtration medium consisting (from top to bottom) of
nitrocellulose, nylon filter, and gel blot paper. RNA that is bound
to the target is captured on the nitrocellulose filter whereas the
non-target bound RNA is captured on the nylon filter. The gel blot
paper is included as a supporting medium for the other filters.
Following filtration, the filter layers are separated, dried and
exposed on a phosphor screen and quantified using a phosphorimaging
system from which. The quantified results can be used to generate
aptamer binding curves from which dissociation constants (K.sub.D)
can be calculated. In a preferred embodiment, the buffered medium
used to perform the binding reactions is 1.times. Dulbecco's PBS
(with Ca.sup.++ and Mg.sup.++) plus 0.1 mg/mL BSA.
Generally, the ability of an aptamer to modulate the functional
activity of a target, i.e., the functional activity of the aptamer,
can be assessed using in vitro and in vivo models, which will vary
depending on the biological function of the target. In some
embodiments, the aptamers of the present invention may inhibit a
known biological function of the target, while in other embodiments
the aptamers of the invention may stimulate a known biological
function of the target. The functional activity of aptamers of the
present invention can be assessed using in vitro and in vivo models
designed to measure a known function of the aptamer target.
The aptamers of the present invention may be routinely adapted for
diagnostic purposes according to any number of techniques employed
by those skilled in the art. Diagnostic utilization may include
both in vivo or in vitro diagnostic applications. Diagnostic agents
need only be able to allow the user to identify the presence of a
given target at a particular locale or concentration. Simply the
ability to form binding pairs with the target may be sufficient to
trigger a positive signal for diagnostic purposes. Those skilled in
the art would also be able to adapt any aptamer by procedures known
in the art to incorporate a labeling tag in order to track the
presence of such ligand. Such a tag could be used in a number of
diagnostic procedures.
Apatamers Having Immunostimulatory Motifs
Recognition of bacterial DNA by the vertebrate immune system is
based on the recognition of unmethylated CG dinucleotides in
particular sequence contexts ("CpG motifs"). One receptor that
recognizes such a motif is Toll-like receptor 9 ("TLR 9"), a member
of a family of Toll-like receptors (.about.10 members) that
participate in the innate immune response by recognizing distinct
microbial components. TLR 9 binds unmethylated oligodeoxynucleotide
("ODN") CpG sequences in a sequence-specific manner. The
recognition of CpG motifs triggers defense mechanisms leading to
innate and ultimately acquired immune responses. For example,
activation of TLR 9 in mice induces activation of antigen
presenting cells, up regulation of MHC class I and II molecules and
expression of important co-stimulatory molecules and cytokines
including IL-12 and IL-23. This activation both directly and
indirectly enhances B and T cell responses, including robust up
regulation of the TH1 cytokine IFN-gamma. Collectively, the
response to CpG sequences leads to: protection against infectious
diseases, improved immune response to vaccines, an effective
response against asthma, and improved antibody-dependent
cell-mediated cytotoxicity. Thus, CpG ODNs can provide protection
against infectious diseases, function as immuno-adjuvants or cancer
therapeutics (monotherapy or in combination with a mAb or other
therapies), and can decrease asthma and allergic response.
Aptamers of the present invention, e.g. MNA aptamers, may comprise
one or more CpG or other immunostimulatory sequence. Such aptamers
can be identified or generated by a variety of strategies using,
e.g., the SELEX.TM. process described herein. In general the
strategies can be divided into two groups. In group one, the
strategies are directed to identifying or generating aptamers
comprising both a CpG motif or other immunostimulatory sequence as
well as a binding site for a target, where the target (hereinafter
"non-CpG target") is a target other than one known to recognize CpG
motifs or other immunostimulatory sequences and known to stimulates
an immune response upon binding to a CpG motif. The first strategy
of this group comprises performing SELEX.TM. to obtain an aptamer
to a specific non-CpG target, using an oligonucleotide pool wherein
a CpG motif has been incorporated into each member of the pool as,
or as part of, a fixed region, e.g., in some embodiments the
randomized region of the pool members comprises a fixed region
having a CpG motif incorporated therein, and identifying an aptamer
comprising a CpG motif. The second strategy of this group comprises
performing SELEX.TM. to obtain an aptamer to a specific non-CpG
target preferably a target and following selection appending a CpG
motif to the 5' and/or 3' end or engineering a CpG motif into a
region, preferably a non-essential region, of the aptamer. The
third strategy of this group comprises performing SELEX.TM. to
obtain an aptamer to a specific non-CpG target, wherein during
synthesis of the pool the molar ratio of the various nucleotides is
biased in one or more nucleotide addition steps so that the
randomized region of each member of the pool is enriched in CpG
motifs, and identifying an aptamer comprising a CpG motif. The
fourth strategy of this group comprises performing SELEX.TM. to
obtain an aptamer to a specific non-CpG target, and identifying an
aptamer comprising a CpG motif. The fifth strategy of this group
comprises performing SELEX.TM. to obtain an aptamer to a specific
non-CpG target and identifying an aptamer which, upon binding,
stimulates an immune response but which does not comprise a CpG
motif.
In group two, the strategies are directed to identifying or
generating aptamers comprising a CpG motif and/or other sequences
that are bound by the receptors for the CpG motifs (e.g., TLR9 or
the other toll-like receptors) and upon binding stimulate an immune
response. The first strategy of this group comprises performing
SELEX.TM. to obtain an aptamer to a target known to bind to CpG
motifs or other immunostimulatory sequences and upon binding
stimulate an immune response using an oligonucleotide pool wherein
a CpG motif has been incorporated into each member of the pool as,
or as part of, a fixed region, e.g., in some embodiments the
randomized region of the pool members comprise a fixed region
having a CpG motif incorporated therein, and identifying an aptamer
comprising a CpG motif. The second strategy of this group comprises
performing SELEX.TM. to obtain an aptamer to a target known to bind
to CpG motifs or other immunostimulatory sequences and upon binding
stimulate an immune response and then appending a CpG motif to the
5' and/or 3' end or engineering a CpG motif into a region,
preferably a non-essential region, of the aptamer. The third
strategy of this group comprises performing SELEX.TM. to obtain an
aptamer to a target known to bind to CpG motifs or other
immunostimulatory sequences and upon binding stimulate an immune
response wherein during synthesis of the pool, the molar ratio of
the various nucleotides is biased in one or more nucleotide
addition steps so that the randomized region of each member of the
pool is enriched in CpG motifs, and identifying an aptamer
comprising a CpG motif. The fourth strategy of this group comprises
performing SELEX.TM. to obtain an aptamer to a target known to bind
to CpG motifs or other immunostimulaory sequences and upon binding
stimulate an immune response and identifying an aptamer comprising
a CpG motif. The fifth strategy of this group comprises performing
SELEX.TM. to obtain an aptamer to a target known to bind to CpG
motifs or other immunostimulatory sequences, and identifying an
aptamer which upon binding, stimulate an immune response but which
does not comprise a CpG motif.
A variety of different classes of CpG motifs have been identified,
each resulting upon recognition in a different cascade of events,
release of cytokines and other molecules, and activation of certain
cell types. See, e.g., CpG Motifs in Bacterial DNA and Their Immune
Effects, Annu. Rev. Immunol. 2002, 20:709-760, incorporated herein
by reference. Additional immunostimulatory motifs are disclosed in
the following U.S. Patents, each of which is incorporated herein by
reference: U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116; U.S.
Pat. No. 6,429,199; U.S. Pat. No. 6,214,806; U.S. Pat. No.
6,653,292; U.S. Pat. No. 6,426,434; U.S. Pat. No. 6,514,948 and
U.S. Pat. No. 6,498,148. Any of these CpG or other
immunostimulatory motifs can be incorporated into an aptamer. The
choice of aptamers is dependent on the disease or disorder to be
treated. Preferred immunostimulatory motifs are as follows (shown
5' to 3' left to right) wherein "r" designates a purine, "y"
designates a pyrimidine, and "X" designates any nucleotide:
AACGTTCGAG (SEQ ID NO:136); AACGTT; ACGT, rCGy; rrCGyy, XCGX,
XXCGXX, and X.sub.1X.sub.2CGY.sub.1Y.sub.2 wherein X.sub.1 is G or
A, X.sub.2 is not C, Y.sub.1 is not G and Y.sub.2 is preferably
T.
In those instances where a CpG motif is incorporated into an
aptamer that binds to a specific target other than a target known
to bind to CpG motifs and upon binding stimulate an immune response
(a "non-CpG target"), the CpG is preferably located in a
non-essential region of the aptamer. Non-essential regions of
aptamers can be identified by site-directed mutagenesis, deletion
analyses and/or substitution analyses. However, any location that
does not significantly interfere with the ability of the aptamer to
bind to the non-CpG target may be used. In addition to being
embedded within the aptamer sequence, the CpG motif may be appended
to either or both of the 5' and 3' ends or otherwise attached to
the aptamer. Any location or means of attachment may be used so
long as the ability of the aptamer to bind to the non-CpG target is
not significantly interfered with.
As used herein, "stimulation of an immune response" can mean either
(1) the induction of a specific response (e.g., induction of a Th1
response) or of the production of certain molecules or (2) the
inhibition or suppression of a specific response (e.g., inhibition
or suppression of the Th2 response) or of certain molecules.
Modulation of Pharmacokinetics and Biodistribution of Aptamer
Therapeutics
It is important that the pharmacokinetic properties for all
oligonucleotide-based therapeutics, including aptamers, be tailored
to match the desired pharmaceutical application. While aptamers
directed against extracellular targets do not suffer from
difficulties associated with intracellular delivery (as is the case
with antisense and RNAi-based therapeutics), such aptamers must
still be able to be distributed to target organs and tissues, and
remain in the body (unmodified) for a period of time consistent
with the desired dosing regimen.
Thus, the present invention provides materials and methods to
affect the pharmacokinetics of aptamer compositions, and, in
particular, the ability to tune aptamer pharmacokinetics. The
tunability of (i.e., the ability to modulate) aptamer
pharmacokinetics is achieved through conjugation of modifying
moieties (e.g., PEG polymers) to the aptamer and/or the
incorporation of modified nucleotides (e.g., 2'-fluoro or
2'-O-methyl) to alter the chemical composition of the nucleic acid.
The ability to tune aptamer pharmacokinetics is used in the
improvement of existing therapeutic applications, or alternatively,
in the development of new therapeutic applications. For example, in
some therapeutic applications, e.g., in anti-neoplastic or acute
care settings where rapid drug clearance or turn-off may be
desired, it is desirable to decrease the residence times of
aptamers in the circulation. Alternatively, in other therapeutic
applications, e.g., maintenance therapies where systemic
circulation of a therapeutic is desired, it may be desirable to
increase the residence times of aptamers in circulation.
In addition, the tunability of aptamer pharmacokinetics is used to
modify the biodistribution of an aptamer therapeutic in a subject.
For example, in some therapeutic applications, it may be desirable
to alter the biodistribution of an aptamer therapeutic in an effort
to target a particular type of tissue or a specific organ (or set
of organs). In these applications, the aptamer therapeutic
preferentially accumulates in a specific tissue or organ(s). In
other therapeutic applications, it may be desirable to target
tissues displaying a cellular marker or a symptom associated with a
given disease, cellular injury or other abnormal pathology, such
that the aptamer therapeutic preferentially accumulates in the
affected tissue. For example, as described in the provisional
application U.S. Ser. No. 60/550,790, filed on Mar. 5, 2004, and
entitled "Controlled Modulation of the Pharmacokinetics and
Biodistribution of Aptamer Therapeutics", and in the
non-provisional application U.S. Ser. No. 11/075,648, filed on Mar.
7, 2005, and entitled "Controlled Modulation of the
Pharmacokinetics and Biodistribution of Aptamer Therapeutics",
PEGylation of an aptamer therapeutic (e.g., PEGylation with a 20
kDa PEG polymer) is used to target inflamed tissues, such that the
PEGylated aptamer therapeutic preferentially accumulates in
inflamed tissue.
To determine the pharmacokinetic and biodistribution profiles of
aptamer therapeutics (e.g., aptamer conjugates or aptamers having
altered chemistries, such as modified nucleotides) a variety of
parameters are monitored. Such parameters include, for example, the
half-life (t.sub.1/2), the plasma clearance (C1), the volume of
distribution (Vss), the area under the concentration-time curve
(AUC), maximum observed serum or plasma concentration (C.sub.max),
and the mean residence time (MRT) of an aptamer composition. As
used herein, the term "AUC" refers to the area under the plot of
the plasma concentration of an aptamer therapeutic versus the time
after aptamer administration. The AUC value is used to estimate the
bioavailability (i.e., the percentage of administered aptamer
therapeutic in the circulation after aptamer administration) and/or
total clearance (C1) (i.e., the rate at which the aptamer
therapeutic is removed from circulation) of a given aptamer
therapeutic. The volume of distribution relates the plasma
concentration of an aptamer therapeutic to the amount of aptamer
present in the body. The larger the Vss, the more an aptamer is
found outside of the plasma (i.e., the more extravasation).
The present invention provides materials and methods to modulate,
in a controlled manner, the pharmacokinetics and biodistribution of
stabilized aptamer compositions, e.g. MNA aptamers, in vivo by
conjugating an aptamer, e.g. an MNA aptamer, to a modulating moiety
such as a small molecule, peptide, or polymer terminal group, or by
incorporating modified nucleotides into an aptamer. As described
herein, conjugation of a modifying moiety and/or altering
nucleotide(s) chemical composition alters fundamental aspects of
aptamer residence time in circulation and distribution to
tissues.
In addition to clearance by nucleases, oligonucleotide therapeutics
are subject to elimination via renal filtration. As such, a
nuclease-resistant oligonucleotide administered intravenously
typically exhibits an in vivo half-life of <10 min, unless
filtration can be blocked. This can be accomplished by either
facilitating rapid distribution out of the blood stream into
tissues or by increasing the apparent molecular weight of the
oligonucleotide above the effective size cut-off for the
glomerulus. Conjugation of small therapeutics to a PEG polymer
(PEGylation), described below, can dramatically lengthen residence
times of aptamers in circulation, thereby decreasing dosing
frequency and enhancing effectiveness against vascular targets.
Aptamers can be conjugated to a variety of modifying moieties, such
as high molecular weight polymers, e.g., PEG; peptides, e.g., Tat
(a 13-amino acid fragment of the HIV Tat protein (Vives, et al.
(1997), J. Biol. Chem. 272(25): 16010-7)), Ant (a 16-amino acid
sequence derived from the third helix of the Drosophila
antennapedia homeotic protein (Pietersz, et al. (2001), Vaccine
19(11-12): 1397-405)) and Arg7 (a short, positively charged
cell-permeating peptides composed of polyarginine (Arg.sub.7)
(Rothbard, et al. (2000), Nat. Med. 6(11): 1253-7; Rothbard, J et
al. (2002), J. Med. Chem. 45(17): 3612-8)); and small molecules,
e.g., lipophilic compounds such as cholesterol. Among the various
conjugates described herein, in vivo properties of aptamers are
altered most profoundly by complexation with PEG groups. For
example, complexation of a mixed 2'F and 2'-OMe modified aptamer
therapeutic with a 20 kDa PEG polymer hinders renal filtration and
promotes aptamer distribution to both healthy and inflamed tissues.
Furthermore, the 20 kDa PEG polymer-aptamer conjugate proves nearly
as effective as a 40 kDa PEG polymer in preventing renal filtration
of aptamers. While one effect of PEGylation is on aptamer
clearance, the prolonged systemic exposure afforded by presence of
the 20 kDa moiety also facilitates distribution of aptamer to
tissues, particularly those of highly perfused organs and those at
the site of inflammation. The aptamer-20 kDa PEG polymer conjugate
directs aptamer distribution to the site of inflammation, such that
the PEGylated aptamer preferentially accumulates in inflamed
tissue. In some instances, the 20 kDa PEGylated aptamer conjugate
is able to access the interior of cells, such as, for example,
kidney cells.
Modified nucleotides can also be used to modulate the plasma
clearance of aptamers. For example, an unconjugated aptamer which
incorporates both 2'-F and 2'-OMe stabilizing chemistries, which is
typical of current generation aptamers as it exhibits a high degree
of nuclease stability in vitro and in vivo, displays rapid loss
from plasma (i.e., rapid plasma clearance) and a rapid distribution
into tissues, primarily into the kidney, when compared to
unmodified aptamer.
Peg-Derivatized Nucleic Acids
As described above, derivatization of nucleic acids with high
molecular weight non-immunogenic polymers has the potential to
alter the pharmacokinetic and pharmacodynamic properties of nucleic
acids making them more effective therapeutic agents. Favorable
changes in activity can include increased resistance to degradation
by nucleases, decreased filtration through the kidneys, decreased
exposure to the immune system, and altered distribution of the
therapeutic through the body.
The aptamer compositions of the invention may be derivatized with
polyalkylene glycol ("PAG") moieties. Examples of PAG-derivatized
nucleic acids are found in U.S. patent application Ser. No.
10/718,833, filed on Nov. 21, 2003, which is herein incorporated by
reference in its entirety. Typical polymers used in the invention
include polyethylene glycol ("PEG"), also known as polyethylene
oxide ("PEO") and polypropylene glycol (including poly isopropylene
glycol). Additionally, random or block copolymers of different
alkylene oxides (e.g., ethylene oxide and propylene oxide) can be
used in many applications. In its most common form, a polyalkylene
glycol, such as PEG, is a linear polymer terminated at each end
with hydroxyl groups:
HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--OH.
This polymer, alpha-, omega-dihydroxylpolyethylene glycol, can also
be represented as HO-PEG-OH, where it is understood that the
-PEG-symbol represents the following structural unit:
--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2--
where n typically ranges from about 4 to about 10,000.
As shown, the PEG molecule is di-functional and is sometimes
referred to as "PEG diol." The terminal portions of the PEG
molecule are relatively non-reactive hydroxyl moieties, the --OH
groups, that can be activated, or converted to functional moieties,
for attachment of the PEG to other compounds at reactive sites on
the compound. Such activated PEG diols are referred to herein as
bi-activated PEGs. For example, the terminal moieties of PEG diol
have been functionalized as active carbonate ester for selective
reaction with amino moieties by substitution of the relatively
non-reactive hydroxyl moieties, --OH, with succinimidyl active
ester moieties from N-hydroxy succinimide.
In many applications, it is desirable to cap the PEG molecule on
one end with an essentially non-reactive moiety so that the PEG
molecule is mono-functional (or mono-activated). In the case of
protein therapeutics which generally display multiple reaction
sites for activated PEGs, bi-functional activated PEGs lead to
extensive cross-linking, yielding poorly functional aggregates. To
generate mono-activated PEGs, one hydroxyl moiety on the terminus
of the PEG diol molecule typically is substituted with non-reactive
methoxy end moiety, --OCH.sub.3. The other, un-capped terminus of
the PEG molecule typically is converted to a reactive end moiety
that can be activated for attachment at a reactive site on a
surface or a molecule such as a protein.
PAGs are polymers which typically have the properties of solubility
in water and in many organic solvents, lack of toxicity, and lack
of immunogenicity. One use of PAGs is to covalently attach the
polymer to insoluble molecules to make the resulting PAG-molecule
"conjugate" soluble. For example, it has been shown that the
water-insoluble drug paclitaxel, when coupled to PEG, becomes
water-soluble. Greenwald, et al., J. Org. Chem., 60:331-336 (1995).
PAG conjugates are often used not only to enhance solubility and
stability but also to prolong the blood circulation half-life of
molecules.
Polyalkylated compounds of the invention are typically between 5
and 80 kDa in size however any size can be used, the choice
dependent on the aptamer and application. Other PAG compounds of
the invention are between 10 and 80 kDa in size. Still other PAG
compounds of the invention are between 10 and 60 kDa in size. For
example, a PAG polymer may be at least 10, 20, 30, 40, 50, 60, or
80 kDa in size. Such polymers can be linear or branched. In some
embodiments the polymers are PEG.
In contrast to biologically-expressed protein therapeutics, nucleic
acid therapeutics are typically chemically synthesized from
activated monomer nucleotides. PEG-nucleic acid conjugates may be
prepared by incorporating the PEG using the same iterative monomer
synthesis. For example, PEGs activated by conversion to a
phosphoramidite form can be incorporated into solid-phase
oligonucleotide synthesis. Alternatively, oligonucleotide synthesis
can be completed with site-specific incorporation of a reactive PEG
attachment site. Most commonly this has been accomplished by
addition of a free primary amine at the 5'-terminus (incorporated
using a modifier phosphoramidite in the last coupling step of solid
phase synthesis). Using this approach, a reactive PEG (e.g., one
which is activated so that it will react and form a bond with an
amine) is combined with the purified oligonucleotide and the
coupling reaction is carried out in solution.
The ability of PEG conjugation to alter the biodistribution of a
therapeutic is related to a number of factors including the
apparent size (e.g., as measured in terms of hydrodynamic radius)
of the conjugate. Larger conjugates (>10 kDa) are known to more
effectively block filtration via the kidney and to consequently
increase the serum half-life of small macromolecules (e.g.,
peptides, antisense oligonucleotides). The ability of PEG
conjugates to block filtration has been shown to increase with PEG
size up to approximately 50 kDa (further increases have minimal
beneficial effect as half life becomes defined by
macrophage-mediated metabolism rather than elimination via the
kidneys).
Production of high molecular weight PEGs (>10 kDa) can be
difficult, inefficient, and expensive. As a route towards the
synthesis of high molecular weight PEG-nucleic acid conjugates,
previous work has been focused towards the generation of higher
molecular weight activated PEGs. One method for generating such
molecules involves the formation of a branched activated PEG in
which two or more PEGs are attached to a central core carrying the
activated group. The terminal portions of these higher molecular
weight PEG molecules, i.e., the relatively non-reactive hydroxyl
(--OH) moieties, can be activated, or converted to functional
moieties, for attachment of one or more of the PEGs to other
compounds at reactive sites on the compound. Branched activated
PEGs will have more than two termini, and in cases where two or
more termini have been activated, such activated higher molecular
weight PEG molecules are referred to herein as, multi-activated
PEGs. In some cases, not all termini in a branch PEG molecule are
activated. In cases where any two termini of a branch PEG molecule
are activated, such PEG molecules are referred to as bi-activated
PEGs. In some cases where only one terminus in a branch PEG
molecule is activated, such PEG molecules are referred to as
mono-activated. As an example of this approach, activated PEG
prepared by the attachment of two monomethoxy PEGs to a lysine core
which is subsequently activated for reaction has been described
(Harris et al., Nature, vol. 2: 214-221, 2003).
The present invention provides another cost effective route to the
synthesis of high molecular weight PEG-nucleic acid (preferably,
aptamer) conjugates including multiply PEGylated nucleic acids. The
present invention also encompasses PEG-linked multimeric
oligonucleotides, e.g., dimerized aptamers. The present invention
also relates to high molecular weight compositions where a PEG
stabilizing moiety is a linker which separates different portions
of an aptamer, e.g., the PEG is conjugated within a single aptamer
sequence, such that the linear arrangement of the high molecular
weight aptamer composition is, e.g., nucleic acid-PEG-nucleic acid
(-PEG-nucleic acid).sub.n where n is greater than or equal to
1.
High molecular weight compositions of the invention include those
having a molecular weight of at least 10 kDa. Compositions
typically have a molecular weight between 10 and 80 kDa in size.
High molecular weight compositions of the invention are at least
10, 20, 30, 40, 50, 60, or 80 kDa in size.
A stabilizing moiety is a molecule, or portion of a molecule, which
improves pharmacokinetic and pharmacodynamic properties of the high
molecular weight aptamer compositions of the invention. In some
cases, a stabilizing moiety is a molecule or portion of a molecule
which brings two or more aptamers, or aptamer domains, into
proximity, or provides decreased overall rotational freedom of the
high molecular weight aptamer compositions of the invention. A
stabilizing moiety can be a polyalkylene glycol, such a
polyethylene glycol, which can be linear or branched, a homopolymer
or a heteropolymer. Other stabilizing moieties include polymers
such as peptide nucleic acids (PNA). Oligonucleotides can also be
stabilizing moieties; such oligonucleotides can include modified
nucleotides, and/or modified linkages, such as phosphorothioates. A
stabilizing moiety can be an integral part of an aptamer
composition, i.e., it is covalently bonded to the aptamer.
Compositions of the invention include high molecular weight aptamer
compositions in which two or more nucleic acid moieties are
covalently conjugated to at least one polyalkylene glycol moiety.
The polyalkylene glycol moieties serve as stabilizing moieties. In
compositions where a polyalkylene glycol moiety is covalently bound
at either end to an aptamer, such that the polyalkylene glycol
joins the nucleic acid moieties together in one molecule, the
polyalkylene glycol is said to be a linking moiety. In such
compositions, the primary structure of the covalent molecule
includes the linear arrangement nucleic acid-PAG-nucleic acid. One
example is a composition having the primary structure nucleic
acid-PEG-nucleic acid. Another example is a linear arrangement of:
nucleic acid-PEG-nucleic acid-PEG-nucleic acid.
To produce the nucleic acid-PEG-nucleic acid conjugate, the nucleic
acid is originally synthesized such that it bears a single reactive
site (e.g., it is mono-activated). In a preferred embodiment, this
reactive site is an amino group introduced at the 5'-terminus by
addition of a modifier phosphoramidite as the last step in solid
phase synthesis of the oligonucleotide. Following deprotection and
purification of the modified oligonucleotide, it is reconstituted
at high concentration in a solution that minimizes spontaneous
hydrolysis of the activated PEG. In a preferred embodiment, the
concentration of oligonucleotide is 1 mM and the reconstituted
solution contains 200 mM NaHCO.sub.3-buffer, pH 8.3. Synthesis of
the conjugate is initiated by slow, step-wise addition of highly
purified bi-functional PEG. In a preferred embodiment, the PEG diol
is activated at both ends (bi-activated) by derivatization with
succinimidyl propionate. Following reaction, the PEG-nucleic acid
conjugate is purified by gel electrophoresis or liquid
chromatography to separate fully-, partially-, and un-conjugated
species. Multiple PAG molecules concatenated (e.g., as random or
block copolymers) or smaller PAG chains can be linked to achieve
various lengths (or molecular weights). Non-PAG linkers can be used
between PAG chains of varying lengths.
The 2'-O-methyl, 2'-fluoro and other modified nucleotide
modifications stabilize the aptamer against nucleases and increase
its half life in vivo. The 3'-3'-dT cap also increases exonuclease
resistance. See, e.g., U.S. Pat. Nos. 5,674,685; 5,668,264;
6,207,816; and 6,229,002, each of which is incorporated by
reference herein in its entirety.
Pag-Derivatization of a Reactive Nucleic Acid
High molecular weight PAG-nucleic acid-PAG conjugates can be
prepared by reaction of a mono-functional activated PEG with a
nucleic acid containing more than one reactive site. In one
embodiment, the nucleic acid is bi-reactive, or bi-activated, and
contains two reactive sites: a 5'-amino group and a 3'-amino group
introduced into the oligonucleotide through conventional
phosphoramidite synthesis, for example: 3'-5'-di-PEGylation as
illustrated in FIG. 13. In alternative embodiments, reactive sites
can be introduced at internal positions, using for example, the
5-position of pyrimidines, the 8-position of purines, or the
2'-position of ribose as sites for attachment of primary amines. In
such embodiments, the nucleic acid can have several activated or
reactive sites and is said to be multiply activated. Following
synthesis and purification, the modified oligonucleotide is
combined with the mono-activated PEG under conditions that promote
selective reaction with the oligonucleotide reactive sites while
minimizing spontaneous hydrolysis. In the preferred embodiment,
monomethoxy-PEG is activated with succinimidyl propionate and the
coupled reaction is carried out at pH 8.3. To drive synthesis of
the bi-substituted PEG, stoichiometric excess PEG is provided
relative to the oligonucleotide. Following reaction, the
PEG-nucleic acid conjugate is purified by gel electrophoresis or
liquid chromatography to separate fully, partially, and
un-conjugated species.
The linking domains can also have one or more polyalkylene glycol
moieties attached thereto. Such PAGs can be of varying lengths and
may be used in appropriate combinations to achieve the desired
molecular weight of the composition.
The effect of a particular linker can be influenced by both its
chemical composition and length. A linker that is too long, too
short, or forms unfavorable steric and/or ionic interactions with
the target will preclude the formation of complex between aptamer
and the target. A linker, which is longer than necessary to span
the distance between nucleic acids, may reduce binding stability by
diminishing the effective concentration of the ligand. Thus, it is
often necessary to optimize linker compositions and lengths in
order to maximize the affinity of an aptamer to a target.
All publications and patent documents cited herein are incorporated
herein by reference as if each such publication or document was
specifically and individually indicated to be incorporated herein
by reference. Citation of publications and patent documents is not
intended as an admission that any is pertinent prior art, nor does
it constitute any admission as to the contents or date of the same.
The invention having now been described by way of written
description, those of skill in the art will recognize that the
invention can be practiced in a variety of embodiments and that the
foregoing description and examples below are for purposes of
illustration and not limitation of the claims that follow.
All publications and patent documents cited herein are incorporated
herein by reference as if each such publication or document was
specifically and individually indicated to be incorporated herein
by reference. Citation of publications and patent documents is not
intended as an admission that any is pertinent prior art, nor does
it constitute any admission as to the contents or date of the same.
The invention having now been described by way of written
description, those of skill in the art will recognize that the
invention can be practiced in a variety of embodiments and that the
foregoing description and examples below are for purposes of
illustration and not limitation of the claims that follow.
EXAMPLES
Example 1
Identification of 5'-Leader Sequences Using the TR-SELEX.TM.
Method
A degenerate DNA library with the following design (shown in the 5'
to 3' direction): T7 Promoter/G.sub.4/degenerate 20
nucleotides/3'-Fixed sequence was synthesized with the following
sequence:
TABLE-US-00003 (ARC 1140, SEQ ID NO 7))
5'TAATACGACTCACTATAGGGGNNNNNNNNNNNNNNNNNNNNACGTAAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT3'.
This library was amplified using the 3'-primer AGCTAGCTTACTGCATCGAC
(SEQ ID NO 104) and the 5'-primer TAATACGACTCACTATAG (SEQ ID NO
105). The double-stranded library was then transcribed using
1.times. Transcription Buffer (HEPES 200 mM, DTT 40 mM, spermidine
2 mM, Triton X-100 0.01%) at 37.degree. C. overnight under the
following conditions: 2'-OMe ATP CTP, UTP, GTP 1 mM each, 2'-OH GTP
30 .mu.M, MgCl.sub.2, 6.5 mM, MnCl.sub.2 2.0 mM, 10% w/v PEG-8000,
1 mM GMP, inorganic pyrophosphatase 0.5 units per 100 .mu.L
reaction, and Y639F/H784A/K378R T7 RNA polymerase 200 nM.
The resultant mixture was then precipitated (isopropanol, sodium
chloride, EDTA), gel-purified (10% PAGE), excised and extracted
from the gel, treated with DNase (RQ1, Promega, Madison Wis.),
reverse-transcribed at 65.degree. C. (Thermoscript, Invitrogen,
Carlsbad, Calif.) using the 3'-primer used for PCR, and diluted
directly into a splinted ligation reaction with the following
oligonucleotides.
5' phosphorylated oligonucleotide encoding a T7 promoter (where p
stands for 5'-phosphorylation):
TABLE-US-00004 pTATAGTGAGTCGTATTA 3' (SEQ ID NO 8)
Splint for ligation:
TABLE-US-00005 5'TAATACGACTCACTATAGGGG 3' (SEQ ID NO 9)
This mixture was heat-denatured, annealed, and then T4 DNA ligase
(NEB, Beverley Mass.) was added followed by incubation at
16.degree. C. overnight. Subsequent to the ligation step, the
reaction was directly diluted into a PCR with the primers already
described to amplify the transcribed sequences for input into the
next round of the SELEX.TM. method. This scheme is presented in
FIG. 2.
After three rounds of TR-SELEX.TM. selection, the library was
cloned using a TOPO TA cloning kit per manufacturer's instructions
(Invitrogen, Carlsbad, Calif.), sequenced, and the statistics of
nucleotide occurrence in the degenerate region were analyzed.
Individual clones were assessed by PAGE-gel analysis for their
ability to template the transcription of large concentrations of
transcript, and the sequences of those that produced the highest
yields of transcript were then utilized in the design of libraries
that were in turn assayed by gel analysis for their ability to
template the transcription of high yields of transcript. FIG. 3
shows the average percentage of nucleotide composition of regions
of the twenty degenerate positions before and after 3 rounds of
TR-SELEX.TM. selection. As indicated by FIG. 3, a strong preference
for G from positions 5 to 13 in the transcript (1 to 9 in the
degenerate region) was transcribed, thereafter no nucleotide is
preferentially transcribed.
The clones discovered by sequencing after 3 Rounds of TR-SELEX.TM.
selection were screened by PAGE-gel analysis for their ability to
transcribe 2'-OMe nucleotides using .about.200 nM template,
1.times. Transcription Buffer (HEPES 200 mM, DTT 40 mM, spermidine
2 mM, Triton X-100 0.01%), 2'-OMe ATP CTP, UTP, GTP at 1 mM each,
2'-OH GTP 30 uM, MgCl.sub.2, 6.5 mM, MnCl.sub.2 2 mM, 10% w/v
PEG-8000, 1 mM GMP, inorganic pyrophosphatase 0.5 units per 100
.mu.L reaction, and Y639F/H784A/K378R mutant T7 RNA polymerase 200
nM, at 37.degree. C. overnight. An example of one clone from Round
3, clone AMX411.D6 gave significantly more MNA transcript, as
visualized by PAGE-gel, when compared to clones from Round 0. The
DNA sequences of the clones generated from Round 3 are listed below
(all sequences listed are in the 5'-3' direction):
TABLE-US-00006 SEQ ID NO 10 > AMX(411)_A1 ARC 1140 Rd 3_411-A1
TAATACGACTCACTATAGGGGGTGGGGCCAATGGCGGGATATACGTAACC
GGTTATACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 11 > AMX(411)_B1 ARC
1140 Rd 3_411-B1 TAATACGACTCACTATAGGGGATGTACATATGTATTCGTGACGTGACCGG
TTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 12 > AMX(411)_C1 ARC
1140 Rd 3_411-C1 TAATACGACTCACTATAGGGGGAGCGGGGAGACGTAGTCATCACGTAGCC
GGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 13 > AMX(411)_D1 ARC
1140 Rd 3_411-D1 TAATACNACTCACTATAGGGGGTGGGGGTGGTGGTGATAACGTAACCGGT
TAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 14 > AMX(411)_E1 ARC
1140 Rd 3_411-E1 TAATACGACTCACTATAGGGGGGTGTCACCAGATATGCCTTGAACGTAAC
CCGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 15 > AMX(411)_F1 ARC
1140 Rd 3_411-F1 TAATACGACTCACTATAGGGGGTAGGGGGCACGCACTAACCAACGTAACC
GGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 16 > AMX(411)_G1 ARC
1140 Rd 3_411-G1 TAATACGACTCACTATAGGGGGAGGGGGTGCTGACCNCAAACA SEQ ID
NO 17 > AMX(411)_H1 ARC 1140 Rd 3_411-H1
TAATACGACTCACTATAGGGGTGGGGCTCGGATGAGACAATACGTAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 18 > AMX(411)_A2 ARC
1140 Rd 3_411-A2 TAATACGACTCACTATAGGGGGGGGTGGGTAGGCGAGCACTCCACGTAAC
CAGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 19 > AMX(411)_B2 ARC
1140 Rd 3_411-B2 TAATACGACTCACTATAGGGGGGAAGGACGAGCAGACGAGCAACGTAACC
TGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 20 > AMX(411)_C2 ARC
1140 Rd 3_411-C2 TAATACGACTCACTATAGGGGGGGGCGGTTAGAGTGTAAGTACCGACGTA
ACCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 21 > AMX(411)_D2
ARC 1140 Rd 3_411-D2
TAATACGACTCACTATAGGGGGGTTGCTGTTAGTAACGCCACGTAACCGG
TTAAACTTGGTCGATGCAGTAAGCTAGCT SEQ ID NO 22 > AMX(411)_E2 ARC
1140 Rd 3_411-E2 TAATACGACTCACTATAGGGGGGCGGGAGAATGTTATATAGTTACGGTAA
CCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 23 > AMX(411)_F2
ARC 1140 Rd 3_411-F2
TAATACGACTCACTATAGGGGAAAGGGGCGGTATGGTACACACGTAACAG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 24 > AMX(411)_G2 ARC
1140 Rd 3_411-G2 TAATACGACTCACTATAGGGGGGACGTGTTAGCATTCCAGAATTCGTAAC
CTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 25 > AMX(411)_H2 ARC
1140 Rd 3_411-H2 TAATACGACTCACTATAGGGGGCGTGGGAGATAGGTTCAAGGACGTACCG
GTTATACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 26 > AMX(411)_A3 ARC
1140 Rd 3_411-A3 TAATACGACTCACTATAGGGGGGCTCCGTGCTATCGTCGGATAACGTAAC
CCGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 27 > AMX(411)_B3 ARC
1140 Rd 3_411-B3 TAATACGACTCACTATAGGGGGGGAGAAGGTCTTAAGGTCGCCAACGTAA
CTGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 28 > AMX(411)_C3 ARC
1140 Rd 3_411-C3 TAATACGACTCACTATAGGGGGGGCATACGAGTTTAGGTGGAGACGTAAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 29 > AMX(411)_D3 ARC
1140 Rd 3_411-D3 TAATACGACTCACTATAGGGGGATGATGACTTCCGCGTTAATACGTTACC
GGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 30 > AMX(411)_E3 ARC
1140 Rd 3_411-E3 TAATACGACTCACTATAGGGGTGGGACGCCGTCTGAGTATAACGTACCCG
GTCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 31 > AMX(411)_G3 ARC 1140 Rd
3_411-G3 TAATACGACTCACTATAGGGGGGGGGGGACGTAATCGGCTATCGTTCACG
TAACCGGTTAAACCCGGGTCGATGCAGTAAAGGGCGA SEQ ID NO 32 > AMX(411)_H3
ARC 1140 Rd 3_411-H3
TAATACGACTCACTATAGGGTGGGACGGGCAGCGTGGATGTAGGACGTAA
CCGGTTAAACGCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 33 > AMX(411)_A4
ARC 1140 Rd 3_411-A4
TAATACGACTCACTATAGGGGGGTTTGTCTGAAGTGAAGCAGAACGTAAC
CGGTTAATCCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 34 > AMX(411)_B4 ARC
1140 Rd 3_411-B4 TAATACGACTCACTATAGGGGGGGAGGGCACATCATCGTATCAAACGTAA
CCAGTTAATCCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 35 > AMX(411)_C4
ARC 1140 Rd 3_411-C4
TAATACGACTCACTATAGGGGAGGCTAGAGGACGCGACAGAACGTAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 36 > AMX(411)_D4 ARC
1140 Rd 3_411-D4 TAATACGACTCACTATAGGGGGCGATCGCGAAGGGATTTCAACGTAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 37 > AMX(411)_E4 ARC
1140 Rd 3_411-E4 TAATACGACTCACTATAGGGGGGTAGGGAAAGATTACGGGGCTACGTAAC
CGGTTATACCTGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 38 > AMX(411)_F4 ARC
1140 Rd 3_411-F4 TAATACGACTCACTATAGGGGTGGCTATGGCTAACACGTAACCGGTTATA
CCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 39 > AMX(411)_G4 ARC 1140 Rd
3_411-G4 TAATACGACTCACTATAGGGGGGGGGCGGTGGCTGTGCAAGCGGAAACGT
AACCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 40 > AMX(411)_H4
ARC 1140 Rd 3_411-H4
TAATACGACTCACTATAGGGGGGTGGGGGCACGGTACTGAGTTACGTTAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 41 > AMX(411)_A5 ARC
1140 Rd 3_411-A5 TAATACGACTCACTATAGGGGGGAGTGGGGACAATTAGAAGATGACGTAA
CCGTCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 42 > AMX(411)_B5 ARC 1140
Rd 3_411-B5 TAATACNACTCACTATAGGGGTGCAGTGAGGAGCGACNAGTACGTTACCG
GTTAAATCCGAGTCGATGCAGTAAGCTAGCT SEQ ID NO 43 > AMX(411)_C5 ARC
1140 Rd 3 411-C5 TAATACNACTCACTATAGGGGGACGGGCACTGTGGATGATTTAACGTTAC
CGGTTAAACCCGAGTCGATGCAGTAAGCTAGCT SEQ ID NO 44 > AMX(411)_D5 ARC
1140 Rd 3_411-D5 TAATACNACTCACTATAGGGGTCGATGCAGTAAGCTAGCT SEQ ID NO
45 > AMX(411)_E5 ARC 1140 Rd 3 411-E5
TAATACNACTCACTATAGGGGGTGATATTGACCTCTAACAGCACGTAACC
GGTTAAACCCGGTCGATGCAGTAAGCTAGCT SEQ ID NO 46 > AMX(411)_F5 ARC
1140 Rd 3_411-F5 TAATACGACTCACTATAGGGGGGGGGGTGCAGAGGATGCATCCAAGCTCG
TAATCGGTGGTCGATGCAGTAAGCTAGCT SEQ ID NO 47 > AMX(411)_G5 ARC
1140 Rd 3_411-G5 TAATACGACTCACTATAGGGGGGGGCGGGTGCTTGTGCCTAATCACGTAA
CCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 48 > AMX(411)_H5
ARC 1140 Rd 3_411-H5
TAATACGACTCACTATAGGGGTTTGGTAATCGAACGTGGAACGCAACCGG
TTTAACCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 49 > AMX(411)_A6 ARC
1140 Rd 3_411-A6 TAATACGACTCACTATAGGGGGGATGGAAGAGGCTTGATATCACGTAACC
GGTTAAACCTGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 50 > AMX(411)_B6 ARC
1140 Rd 3_411-B6 TAATACGACTCACTATAGGGGGTTATACTAACTCTGTACACAACGTAACC
GGCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 51 > AMX(411)_C6 ARC 1140
Rd 3_411-C6 TAATACGACTCACTATAGGGGTATAGGGGGGGTATCGGTGTACGTAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 52 > AMX(411)_D6 ARC
1140 Rd 3_411-D6 TAATACGACTCACTATAGGGGAGTACAATAAGGTTCCGAGAACGCGACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 53 > AMX(411)_E6 ARC
1140 Rd 3_411-E6 TAATACGACTCACTATAGGGGTGCGCGGTTACAAGGCAACATACGTAACC
GGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 54 > AMX(411)_F6 ARC
1140 Rd 3_411-F6 TAATACNACTCACTATAGGGGGGACGGGGTGACAAAGTGTCNAACGTAAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 55 > AMX(411)_G6 ARC
1140 Rd 3_411-G6 TAATACGACTCACTATAGGGGAGACGGCGGTACAAGTCCATATGTAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 56 > AMX(411)_H6 ARC
1140 Rd 3_411-H6 TAATACGACTCACTATAGGGGAGTGGGGGCTTCTCGTTGCCACGTAACCG
CTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 57 > AMX(423)_A7 ARC
1140 R3_423-A7 TAATACGACTCACTATAGGGGGGCTGAGCGTGTTTGAGGGACCACGTTAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 58 > AMX(423)_B7 ARC
1140 R3_423-B7 TAATACGACTCACTATAGGGGGGTGGGCGCAATGAAAAGTTGGGCGTAAC
CGGTTCAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 59 > AMX(423)_C7 ARC
1140 R3_423-C7 TAATACGACTCACTATAGGGGGTAGTGAAGTAAGGCAGTGTTACGTAACC
GGTGAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 60 > AMX(423)_D7 ARC
1140 R3_423-D7 TAATACGACTCACTATAGGGGGGAGGGTGGGCTAGAACACACAACGTAAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT
SEQ ID NO 61 > AMX(423)_E7 ARC 1140 R3_423-E7
TAATACGACTCACTATAGGGGGGGAGAGAGGCGGTTACGTAGGGACGTTA
CCGATTGAACTCAGGTCGATGCAGTAAGCTAGCT SEQ ID NO 62 > AMX(423)_F7
ARC 1140 R3 423-F7
TAATACGACTCACTATAGGGGGGGGGGGCGAATAGGTAGGGCGACGAACG
TTACCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 63 > AMX(423)_G7
ARC 1140 R3_423-G7
TAATACGACTCACTATAGGGGGAGAGGAGGTCCGGCTAGACAACGTAACC
GGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 64 > AMX(423)_H7 ARC
1140 R3_423-H7 TAATACGACTCACTATAGGGGGGAGGACGGGTCGTACTGTTAAACCTGGG
TCGATGCAGTAAGCTAGCT SEQ ID NO 65 > AMX(423)_B8 ARC 1140
R3_423-B8 TAATACGACTCACTATAGGGGGCGCAACAACGGGAAGTATACGTAACCGG
TTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 66 > AMX(423)_C8 ARC
1140 R3_423-C8 TAATACGACTCACTATAGGGGGAAGGAACACGCACATGCATAACGTAACT
GGTTGACCCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 67 > AMX(423)_D8 ARC
1140 R3_423-D8 TAATACGACTCACTATAGGGGAGTGGGGAGTACTGTGGACAACGTGACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 68 > AMX(423)_E8 ARC
1140 R3_423-E8 TAATACGACTCACTATAGGGGTCGATGCAGTAAGCTAGCT SEQ ID NO
69 > AMX(423)_F8 ARC 1140 R3_423-F8
TAATACGACTCACTATAGGGGGGGGGGCTAGGGCGGTCGGATCGGACGTA
ACCAGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 70 > AMX(423)_G8
ARC 1140 R3_423-G8
TAATACGACTCACTATAGGGGGGGTGGGGGTTGCTACATGCCCTCGTAAC
CGGTTAAGCCCAGGTCGATGCAGTAAGCTAGCT SEQ ID NO 71 > AMX(423)_H8 ARC
1140 R3_423-H8 TAATACGACTCACTATAGGGGGGTGGCGACGATGGAGAGAATAACGTAAT
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 72 > AMX(423)_A9 ARC
1140 R3_423-A9 TAATACGACTCACTATAGGGGGTAGGCGGGCCTCATCAACAACGCAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 73 > AMX(423)_B9 ARC
1140 R3_423-B9 TAATACGACTCACTATAGGGGGTGGCTGGTAAGGACACAAACACGTAACT
CGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 74 > AMX(423)_C9 ARC
1140 R3_423-C9 TAATACGACTCACTATAGGGGGGCGGGCAGCGCTTATAGATCCACGTAAC
CGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 75 > AMX(423)_D9 ARC
1140 R3_423-D9 TAATACGACTCACTATAGGGGGGGGGTATCTGCGGTTAGGCTATCGACGT
ACCCAGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 76 > AMX(423)_F9
ARC 1140 R3_423-F9
TAATACGACTCACTATAGGGGGGGTAGGGGACATCATAGGTATACGTAAC
CGGTTAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 77 > AMX(423)_H9 ARC
1140 R3_423-H9 TAATACGACTCACTATAGGGGCGCGTGCGTGTATCCATTAAACGTGACTG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 78 > AMX(423)_A10 ARC
1140 R3_423-A10 TAATACGACTCACTATAGGGGGGGGAGCGTGGATCTTGAGTGTATACGT
AACCGGTTAAACCCGGTCGATGCAGTAAGCTAGCT SEQ ID NO 79 > AMX(423)_B10
ARC 1140 R3_423-B10
TAATACGACTCACTATAGGGGATGGAGAGGAGTGTACGCATATACAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 80 > AMX(423)_C10 ARC
1140 R3_423-C10 TAATACGACTCACTATAGGGGCGGGTGGTCGCGATGGTTAACGTAACTGG
TTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 81 > AMX(423)_D10 ARC
1140 R3_423-D10 TAATACGACTCACTATAGGGGGGGGGGGGGACGTTAGCTTCTCTGTATTT
ACGTAACCGGTTAAGCCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 82 >
AMX(423)_E10 ARC 1140 R3_423-E10
TAATACGACTCACTATAGGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 83 >
AMX(423)_F10 ARC 1140 R3 423-F10
TAATACGACTCACTATAGGGGGGATGGAGTGGGTGCAAATAANACGTAAC
TGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 84 > AMX(423)_G10
ARC 1140 R3_423-G10
TAATACGACTCACTATAGGGGAGNGTGAGGGGTGAATANTAANGTAANCN
GTTAAACCTGGGTCGATGNNNTANNCTNGNT SEQ ID NO 85 > AMX(423)_H10 ARC
1140 R3_423-H10 NAATNNGACTCACAANAGGGGTCGATGCAGTAAGCTAGCT SEQ ID NO
86 > AMX(423)_A11 ARC 1140 R3_423-A11
TAATACGACTCACTATAGGGGGGGGTGACGTACGGATCTAAGTAACGTAA
CCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 87 > AMX(423)_B11
ARC 1140 R3_423-B11
TAATACGACTCACTATAGGGGAGGGACAGACACTTTGTAGACGTAACCAG
TTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 88 > AMX(423)_C11 ARC
1140 R3_423-C11 TAATACGACTCACTATAGGGGGGGGACTTGGCACTACGTAACAACGTAAC
CGCTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 89 > AMX(423)_D11
ARC 1140 R3_423-D11
TAATACGACTCACTATAGGGGGGGGGGCCTCTCGACCAAAAGCCCAACGT
AACCGGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 90 > AMX(423)_E11
ARC 1140 R3_423-E11
TAATACNACTCACTATAGGGGGGGGGGGATAGTCATGACTGATAAAACGT
AACTGTTGAGCCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 91 > AMX(423)_F11
ARC 1140 R3_423-F11
TAATACGACTCACTATAGGGGACAGTGCTAGTGGAATAGCAACGTAACCA
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 92 > AMX(423)_G11 ARC
1140 R3_423-G11 TAATACGACTCACTATAGGGGACGACCACTATACTCCGAGAACGTAACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 93 > AMX(423)_H11 ARC
1140 R3_423-H11 TAATACGACTCACTATAGGGGGATGGAGGCGTAGTGTAGTCAACGTTACC
GGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 94 > AMX(423)_A12 ARC
1140 R3_423-A12 TAATACGACTCACTATAGGGGGGAGGTATAGATGGAATGGTTATGTAACC
TGTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 95 > AMX(423)_B12 ARC
1140 R3_423-B12 TAATACGACTCACTATAGGGGTGGGGAGGACCACTTAGATAACGTCACCG
GTTAAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 96 > AMX(423)_C12 ARC
1140 R3_423-C12 TAATACGACTCACTATAGGGGGGATAGGGGCGAGAGAGTCACAACGTAAC
CGGTTAATCCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 97 > AMX(423)_E12
ARC 1140 R3_423-E12
TAATACGACTCACTATAGGGGGGGGATGGCCGAATCATAAAATAACGTAA
CCGTTAGACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 98 > AMX(423)_F12
ARC 1140 R3_423-F12
TAATACGACTCACTATAGGGGGCGATTGCTGAGTCAGTTCGTAATCGGTT
AAACCCGGGTCGATGCAGTAAGCTAGCT SEQ ID NO 99 > AMX(423)_G12 ARC
1140 R3_423-G12 TAATACGACTCACTATAGGGGGGGGAGGATCCGAAACACAGGGATCCGTA
ACCGGTTAAAGCCGGGTCGATGCAGTAAGCTAGCT
Example 2
Libraries Incorporating Leader Sequences Identified by the
TR-SELEX.TM. Method
The identified 5'-leader sequence elements (the first 10
nucleotides of the degenerate region) from higher 2'-modified
transcript-yielding clones identified using TR-SELEX.TM. selection
as described in Example 1 were utilized to design libraries which
incorporate the leader sequence elements into the 5'-fixed region,
with the goal of promoting an increase in transcript yield
containing 2'-modified nucleotides. In one embodiment, the design
strategy incorporates the first 14 nucleotides of the identified
clones (the 4 guanosines comprising the 5' fixed region plus the
first 10 nucleotides of the degenerate region) as the 5'-leader
sequence immediately followed by an additional 6-8 fixed
nucleotides to facilitate subsequent PCR amplification, immediately
followed by a degenerate region 30-40 nucleotides in length,
immediately followed by a 3'-fixed region to also facilitate
subsequent PCR amplification.
Examples of the DNA sequences of the libraries designed which
incorporate the identified leader sequence elements are listed
below.
For each of the sequences of the libraries of DNA transcription
templates listed below, the 5'-leader sequence element is shown
underlined, and all sequences are in the 5'-3' direction.
TABLE-US-00007 ARC 2118 (SEQ ID NO 3)
TAATACGACTCACTATAGGGGAGTACAATAACGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2119 (SEQ ID NO 4)
TAATACGACTCACTATAGGGGGTGATATTGACGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2120 (SEQ ID NO 5)
TAATACGACTCACTATAGGGGTGCGCGGTTACGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2121 (SEQ ID NO 6)
TAATACGACTCACTATAGGGGGAGGGGGTGCCGTTCTCGNNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG
A control DNA transcription templates without a leader sequence is
listed below, in the 5'-3' direction.
TABLE-US-00008 (ARC2117, SEQ ID NO 106)
TAATACGACTCACTATAGGGGAGAGGAGAGAACGTTCTCGNNNNNNNNNN
NNNNNNNNNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG
To test whether the newly designed libraries promote an increased
yield of transcripts containing 2'-O-methyl nucleotides, the
libraries were transcribed using two different modified T7 RNA
polymerases for comparison, the Y639F/H784A/K378R mutant T7 RNA
polymerase, and the Y639L/H784A/K378R mutant polymerase
(transcription reaction mixtures without polymerase was used as a
negative control), in a transcription mixture containing .about.200
nM template, 1.times. transcription buffer (HEPES 200 mM, DTT 40
mM, spermidine 2 mM, Triton X-100 0.01%), 2'-OMe ATP, CTP, UTP, and
GTP 1 mM each, 2'-OH GTP at 30 uM, MgCl.sub.2 6.5 mM, MnCl.sub.2
2.0 mM, PEG-8000 w/v 10%, GMP 1 mM, Y639F/H784A/K378R mutant T7 RNA
polymerase or Y639L/H784A/K378R mutant T7 RNA polymerase 200 nM,
inorganic pyrophosphatase 5 units/mL, at 37.degree. C. overnight.
Transcript yield for each condition was assayed by PAGE-gel
analysis using 200 uL of reaction mixture, and transcript yield for
each condition was quantitated from UV-shadowing of the PAGE-gel
analysis using ImageQuant version 5.2 software (Molecular
Dynamics).
FIG. 4 summarizes the quantitated results of the PAGE-gel analysis,
showing the fold-increase of transcript yield with both
Y639F/H784A/K378R ("FAR") and Y639L/H784A/K378R ("LAR") mutant T7
RNA polymerases relative to the no polymerase negative control. As
can be seen in FIG. 4, a significant improvement in the yield of
fully 2'-OMe containing transcripts was seen when the
Y639L/H784A/K378R mutant T7 RNA polymerase was used to transcribe
the new libraries incorporating the new leader sequence elements as
compared to the Y639F/H784A/K378R mutant polymerase. Notably,
ARC2118, ARC2119, ARC2120 gave significantly higher yields of
2'-OMe containing transcripts when combined with the
Y639L/H784A/K378R mutant T7 RNA polymerase as compared to the
Y639F/H784A/K378R mutant T7 RNA polymerase. An increase in
transcript yield by using the Y639L/H784A/K378R mutant T7 RNA
polymerase was also seen with ARC2117, a library formerly designed
which lacks the newly identified leader sequence elements, known to
transcribe under the given conditions with the Y639F/H794A/K378R
mutant polymerase, which was used as a control. These results
indicate that the yields of 2'-OMe-containing transcript may be
increased by utilizing the Y639L/H784A/K378R mutant T7 RNA
polymerase as compared to the Y639F/H784A/K378R mutant T7 RNA
polymerase. In addition, several of the new libraries (ARC2118 and
ARC2119) incorporating the leader sequence elements identified
through the TR-SELEX.TM. method also gave higher yields of 2'-OMe
containing transcripts than the control library, ARC2117, when
using the Y639L/H784A/K378R mutant T7 RNA polymerase, indicating
that an improvement in the yield of 2'-OMe containing transcript
can be achieved by utilizing the Y639L/H784A/K378R mutant in
combination with the particular newly identified leader sequences
of the present invention.
Example 3
Polymerase Expression and Purification
Mutant T7 RNA polymerase, for use in the methods of the invention
may be prepared as follows. T7 RNA polymerase (nucleic acid and
amino acid sequence shown in FIGS. 5A and 5B respectively and
described in Bull, J. J et al., J. Mol. Evol., 57 (3), 241-248
(2003) may be mutated to result in the LA mutant (Y639L/H784A), the
LAR mutant (Y639L/H784A/K378R), the LLA mutant (P266L/Y639L/H784A)
or the LLAR mutant P266L/Y639L/H784A/K378R). T7 RNA polymerase may
be comprised in an expression vector (an example of a T7 RNA
polymerase expression vector is described in U.S. Pat. No.
5,869,320 herein incorporated by reference in its entirety) or may
be inserted into an expression vector following mutagenesis. The
mutated T7 RNA polymerase may be engineered to optionally comprise
a His-tag for ease during protein purification.
Complementary oligonucleotide sequences that contain the Leucine
mutation for position 639 (agtcatgacgctggctCTGgggtccaaagagttcg (SEQ
ID NO 107 and gaactctttggacccCAGagccagcgtcatgact (SEQ ID NO 108)
may be synthesized. Complementary oligonucleotide sequences
(ggctggcatctctcTgatgttccaaccttgc (SEQ ID NO 109) and
gcaaggttggaacatcAgagagatgccagcc (SEQ ID NO 110) for P266L mutation
may be synthesized. Complementary oligonucleotide sequences
(cgctcctaactttgtaGCcagccaagacggtagc (SEQ ID NO 111) and
gctaccgtcttggctgGCtacaaagttaggagcg (SEQ ID NO 112)) for H784A
mutation may be synthesized. Complementary oligonucleotide
sequences (gctctcaccgcgtggaGacgtgctgccgctgct (SEQ ID NO 113) and
agcagcggcagcacgtCtccacgcggtgagagc (SEQ ID NO 114)) for K378R
mutation may be synthesized. Site-directed mutagenesis may be
performed using QuikChanges.RTM. Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, Calif.) according to the manufacturer's
instructions to result in nucleic acid sequences (FIG. 6) encoding
mutant polymerases having the above indicated combination of
mutations. The resulting nucleic acid sequence encoding a mutant
polymerase of the invention may be inserted into the desired
expression vector using standard techniques for expression and
purification.
Expression and Purification
The expression vector comprising the mutant T7 polymerase nucleic
acid sequence is is transformed int BL21 (DE3) competent cells
(Stratagene, Calif.) and incubated on ice for 20 min. Heat shock is
performed by putting the tube in 42.degree. C. for 2 min. After
putting the tube on ice for 1 minute, 1 ml L broth ("LB") is added
and incubated in 37.degree. C. shaker for 45 min. 100 ul of culture
liquid is plated on LB+Amp agar plate and incubated at 37.degree.
C. overnight.
A single colony from the overnight cultured plate is inoculated
into 100 ml LB-Amp+ (150 ug/ml), 37.degree. C. overnight. On the
second day, two 4-liter flasks containing 2 liters of pre-warmed
LB+Amp are inoculated with 50 ml of overnight culture and grown at
37.degree. C. until OD600 reaches between 0.6-0.8. 200 ul of 1M
IPTG is added to each 2 L cell culture with final concentration of
100 uM and grow for another 3 hrs at 37.degree. C. The cells are
pelleted by spinning at 5000 rpm for 10 min. Cells are resuspended
in 200 ml lysis buffer (Lysis buffer: 50 mM Tris-Cl, pH 8.0, 100 mM
NaCl, 5% Glycerol, 1 mM imidazole, betamercaptoethanol ("BME") 5
mM) and divided into 6 conical 50 ml tubes. The cells are sonicated
at power level 8, 3.times.30'' for each tube and then bacterial
debris is spun down at 11,000 rpm for 60 min and the supernatant
filtered through 0.22 uM filter. Imidazole is added to the filtrate
to a final concentration of 10 mM.
The filtrate is loaded onto a 5 ml Ni-NTA column (GE Healthcare
Bio-Sciences, NJ) with sample pump. The column is washed with 10
column volumes (CV) of buffer A (Buffer A: 50 mM Tris-Cl, pH 8.0,
100 mM NaCl, 5% Glycerol, 10 mM imidazole, BME 10 mM) containing 20
mM imidazole. The column is then washed with 10 CV of buffer with a
linear gradient of imidazole concentration from 40 mM to 70 mM in
buffer A. The protein is eluted with 6 CV of Buffer B (Buffer B: 50
mM Tris-Cl, pH 8.0, 100 mM NaCl, 5% Glycerol, 250 mM imidazole, BME
10 mM). After checking the collection fractions with 5 .mu.l of
sample on 4-12% SDS-PAGE, all the fractions of interest are
combined and dialyzed (dialysis tubing: Spectrum Spectra/por
Molecular porous membrane (VWR) MWCO: 12-14000) in 1 L of dialysis
buffer (Dialysis buffer: 50 mM Tris-Cl, pH 7.9, 100 mM NaCl, 50%
Glycerol, 0.1 mM EDTA, 0.1% Triton X-100, BME 20 mM) overnight. The
dialysis buffer is changed after 12 hours and dialysis is carried
out for an additional 4 hours. The concentration of T7 RNA
polymerase is measured using the Bradford assay as described in
Bradford, M. M. (1976) Anal. Biochem. 72, 248.
Example 4
Transcription incorporating 100% 2'-O-methyl nucleotides
Example 4A
2'-O-methyl transcription without 2'-OH GTP
An experiment was performed to test the sensitivity of
Y639L/H784A/K378R mutant polymerase to the concentration of 2'-OH
GTP by using a titration of 2'-OH GTP.
ARC2118 and ARC2119, two libraries incorporating the new leader
sequence elements identified through TR-SELEX.TM. selection
(described in Example 1), which showed high transcript yields when
used with the Y639L/H784A/K378R mutant T7 RNA polymerase (see
Example 2), were used to test the sensitivity of transcription of
the Y639L/H784A/K378R mutant T7 RNA polymerase to the concentration
of 2'-OH GTP. Transcriptions were performed using a titration of
2'-OH GTP (0-160 uM) with 1.times. transcription buffer (HEPES 200
mM, DTT 40 mM, spermidine 2 mM, Triton X-100 0.01%), .about.200 nM
template, 2'-OMe ATP, CTP, UTP, and GTP 1 mM each, MgCl.sub.2 6.5
mM, MnCl.sub.2 2.0 mM, PEG-8000 w/v 10%, GMP 1 mM, inorganic
pyrophosphatiase 5 units/mL, Y639L/H784A/K378R mutant T7 RNA
polymerase 200 nM, at 37.degree. C. overnight.
Transcript yield under each condition was assayed by PAGE-gel
analysis using 200 uL of reaction mixture, and transcript yield for
each condition was quantitated from UV-shadowing of the PAGE-gel
analysis using ImageQuant version 5.2 software (Molecular
Dynamics). FIG. 7 summarizes the quantitated results of the
PAGE-gel analysis, showing the fold-increase of transcript yield
with of each condition relative to the background. As can be seen
in FIG. 7, ARC2118 and ARC2119 transcribed with Y639L/H784A/K378R
under all conditions, including no 2'-OH GTP, and the yield in the
absence of 2'-OH GTP was comparable to transcription yield where
2'-OH GTP was included in the reaction mixture. These results
indicate that the Y639L/H784A/K378R mutant T7 RNA polymerase does
not require the presence of 2'-OH GTP for increased transcript
yield, as opposed to the Y639F/H784A/K378R mutant T7 RNA
polymerase, which requires 2'-OH GTP for transcription (data not
shown).
An experiment was subsequently performed to determine the optimal
transcription conditions to be used with the Y639L/H784A/K378R
mutant T7 RNA polymerase when combined with the leader sequences
identified by TR-SELEX.TM. selection, (described in Example 1).
ARC2119, a library incorporating the new leader sequence elements
identified through TR-SELEX.TM. selection which showed
significantly higher transcript yield when used with the
Y639L/H784A/K378R mutant T7 RNA polymerase (see Example 2) was used
to test the effect of varying the 2'-OMe NTP, magnesium and
manganese concentrations on transcript yield.
Transcriptions were performed using 1.times. transcription buffer
(HEPES 200 mM, DTT 40 mM, spermidine 2 mM, Triton X-100 0.01%),
.about.200 nM template, 2'-OMe ATP, CTP, UTP, and GTP (0.5 mM, 1
mM, 1.5 mM, and 2 mM each), MgCl.sub.2 (5 mM, 6.5 mM, 8 mM, and 9.5
mM), MnCl.sub.2 (1.5 mM, 2 mM, 2.5 mM, 3 mM), PEG-8000 w/v 10%, GMP
1 mM, inorganic pyrophosphatase 5 units/mL, Y639L/H784A/K378R
mutant T7 RNA polymerase 200 nM, at 37.degree. C. overnight.
Transcript yield under each condition was assayed by PAGE-gel
analysis using 200 uL of reaction mixture, and transcript yield for
each condition was quantitated from UV-shadowing of the PAGE-gel
analysis using ImageQuant version 5.2 software (Molecular
Dynamics). FIG. 8 summarizes the quantitated results of the
PAGE-gel analysis, showing the fold-increase of transcript yield
with of each condition relative to background. Based on the cost of
2'-OMe NTPs, and the results of this experiment, 1.5 mM each 2'-OMe
NTP (and 8 mM MgCl.sub.2, 2.5 mM MnCl.sub.2) was adopted as the
preferred conditions to use with the leader sequences and the
Y639L/H784A/K378R mutant T7 RNA polymerase of the present
invention.
Example 4B
Fidelity and Bias of MNA Transcription Using Y639L/H784A/K378R
Mutant T7 RNA Polymerase
Additional experiments were performed to assess the fidelity and
bias of MNA transcription using the Y639L/H784A/K378R mutant T7 RNA
polymerase and no 2'-OH GTP. To test fidelity, a single cloned
sequence identified by TR-SELEX.TM. selection (described in Example
1) was amplified by PCR, used to program a MNA transcription using
the Y639L/H784A/K378R polymerase and no 2'-OH GTP, purified by
PAGE, remaining DNA template was digested using RQ1 DNase (the
absence of DNA template was then assayed by PCR) and the
transcribed material was reverse-transcribed (Thermoscript,
Invitrogen, Carlsbad, Calif.) and then amplified by PCR. This PCR
product was sequenced and the statistics of deletions, insertions
and substitutions was then calculated. Of the 1300 bases sequenced
in this experiment, no deletions and insertions were observed, and
three substitutions were observed (see FIG. 9). These numbers
suggest that the sequence information encoded within a
30-nucleotide degenerate region would have a 93% chance of being
faithfully transmitted to the next round of SELEX.TM., this number
is so high that it exceeds that measured for wild-type RNA.
To test for nucleotide bias, library ARC2118 was transcribed under
the following conditions: HEPES 200 mM, DTT 40 mM, spermidine 2 mM,
Triton X-100 0.01%, .about.200 nM template, 2'-OMe ATP, CTP, UTP,
and GTP 1 mM each (no 2'-OH GTP), MgCl.sub.2 (6.5 mM), MnCl.sub.2
(2 mM), PEG-8000 w/v 10%, GMP 1 mM, inorganic pyrophosphatase 5
units/mL, Y639L/H784A/K378R mutant T7 RNA polymerase 200 nM, at
37.degree. C. overnight, purified by PAGE, the remaining DNA
template was digested using RQ1 DNase (the absence of DNA template
was then assayed by PCR) and the transcribed material was
reverse-transcribed and amplified using PCR before cloning and
sequencing. 48 clones from the amplified library and 48 clones from
the starting library were sequenced. The statistics of nucleotide
occurrence in the degenerate region were examined to see if bias
occurred. As indicated by FIG. 10, the percentage of nucleotide
composition after transcription was very similar to the percentage
of nucleotide composition of the starting library in which the
percentage of each nucleotide (A, T, C and G) was approximately
equal, indicating that no nucleotide bias occurs with the
Y639L/H784A/K378R mutant T7 RNA polymerase is used for
transcription.
Example 4C
Comparison of Transcriptional Yield with Various Leader
Sequences
Templates 1 to 4:
To compare transcriptional yields using Y639L/H784A/K378R mutant T7
RNA polymerase with multiple different leader sequences, 4
templates comprising varying ratios of purines to pyrimidines in
the leader sequence (positions 1 to 14 in SEQ ID NOs 126 to 129
below), were synthesized with different constant regions. The DNA
templates were synthesized using an ABI EXPEDITE.TM. (Applied
Biosystems, Foster City, Calif.) DNA synthesizer, and deprotected
by standard methods. The sequences (shown in the 5' to 3') are as
follows:
TABLE-US-00009 Template 1 GGGAGAATTCCGACCAGAAGCTTNNNNNNNNNNN (SEQ
ID NO 126) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNCATAT GTGCGTCTACATGGATCCTCA
Template 2 GGGAGAGCGGAAGCCGTGCTGGGGCCNNNNNNNN (SEQ ID NO 127)
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCA TAACCCAGAGGTCGATGGATC Template 3
GGGAGAGACAAGCTTGGGTCNNNNNNNNNNNNNN (SEQ ID NO 128)
NNNNNNNNNNNNNNNNNNNNNNNNNNAGAAGAGA AAGAGAAGTTAATTAAGGATCCTCAG
Template 4 GGGAGAATTCCGACCACAAGNNNNNNNNNNNNNN (SEQ ID NO 129)
NNNNNNNNNNNNNNNNNNNNNNNNNNCATATGTG CGTCTACATGGATCCTCA
The templates were amplified with their respective primers as
indicated in below:
TABLE-US-00010 Template 1: 5' primer
TAATACGACTCACTATAGGGAGAATTCCGACCAG (SEQ ID NO 130) AAGCTT 3' primer
TGAGGATCCATGTAGACGCACATATG (SEQ ID NO 131) Template 2: 5' primer
TAATACGACTCACTATAGGGAGAGCGGAAGCCGT (SEQ ID NO 149) GCTGGGGCC 3'
primer GATCCATCGACCTCTGGGTTATG (SEQ ID NO 132) Template 3: 5'
primer TAATACGACTCACTATAGGGAGAGACAAGCTTGG (SEQ ID NO 133) GTC 3'
primer CTGAGGATCCTTAATTAACTTCTCTTTCTCTTCT (SEQ ID NO 134) Template
4: 5' primer TAATACGACTCACTATAGGGAGAATTCCGACCAC (SEQ ID NO 135) AAG
3' primer TGAGGATCCATGTAGACGCACATATG (SEQ ID NO 148)
The templates were used in a 15 mL in vitro transcription reaction
with T7 RNA polymerase (Y639L/H784A/K378R). Transcriptions were
done using 200 mM Hepes, 40 mM DTT, 2 mM spermidine, 0.01% Triton
X-100, 10% PEG-8000, 8 mM MgCl.sub.2, 2.5 mM MnCl.sub.2, 1.5 mM
mCTP, 1.5 mM mUTP, 1.5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01
units/.mu.L inorganic pyrophosphatase, and .about.9 .mu.g/ml T7
polymerase (Y639L/H784A/K378R) and 0.2 .mu.M template DNA. The RNA
was precipitated and purified on 10% denaturing PAGE. The RNA was
eluted from the gel in 300 mM NaOAc, 20 mM EDTA overnight,
precipitated and quantitated with a UV Spec. The yields did not
differ greatly between the four leader sequences tested and are
shown in Table 1A below.
TABLE-US-00011 TABLE 1A Pool Yield (nmoles) Pool 1 18.7 Pool 2 17.4
Pool 3 20.2 Pool 4 27.8
Templates 5 to 8
As for Templates 1 to 4 above, transcriptional yields for multiple
leader sequences was assessed. Four templates were synthesized with
different constant regions. The DNA templates were synthesized
using an ABI EXPEDITE.TM. (Applied Biosystems, Foster City, Calif.)
DNA synthesizer, and deprotected by standard methods. The sequences
(shown in the 5' to 3' direction) are as follows:
TABLE-US-00012 Template 5 GGGCCTTGTAGCGTGCATTCTTGNNNNNNNNNNN (SEQ
ID NO 138) NNNNNNNNNNNNNNNNNNNCTAACATACTCCGAA TCTGTCGAA Template 6
GGAGCCTTCCTCCGGANNNNNNNNNNNNNNNNNN (SEQ ID NO 139)
NNNNNNNNNNNNNNNNNNNNNNTCCGGTTTCCCG AGCTT Template 7
GGGAGACAAGAATAAACGCTCAANNNNNNNNNNN (SEQ ID NO 140)
NNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTCGA CAGGAGGCTCACAACAGGC Template 8
GGGGAGTACAATAACCAGACATNNNNNNNNNNNN (SEQ ID NO 150)
NNNNNNNNNNNNNNNNNNGGATCGTTACGACTAG CATCGATG
The templates were amplified with their respective primers:
TABLE-US-00013 Template 5 5' primer
TAATACGACTCACTATAGGGCCTTGTAGCGTGCA (SEQ ID NO 151) TTCTTG 3' primer
TTCGACAGATTCGGAGTATGTTAG (SEQ ID NO 141) Template 6 5' primer
TAATACGACTCACTATAGGAGCCTTCCTCCGGA (SEQ ID NO 142) 3' primer
AAGCTCGGGAAACCGGA (SEQ ID NO 143) Template 7 5' primer
TAATACGACTCACTATAGGGAGACAAGAATAAAC (SEQ ID NO 144) GCTCAA 3' primer
GCCTGTTGTGAGCCTCCTGTCGAA (SEQ ID NO 145) Template 8 5' primer
TAATACGACTCACTATAGGGGAGTACAATAACCA (SEQ ID NO 146) GACAT 3' primer
CATCGATGCTAGTCGTAACGATCC (SEQ ID NO 147)
The templates were then used for a 0.5 mL in vitro transcription
with T7 RNA polymerase (Y639L/H784A/K378R). Transcriptions were
done using 200 mM Hepes, 40 mM DTT, 2 mM spermidine, 0.01% Triton
X-100, 10% PEG-8000, 8 mM MgCl.sub.2, 2.5 mM MnCl.sub.2, 1.5 mM
mCTP, 1.5 mM mUTP, 1.5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01
units/.mu.L inorganic pyrophosphatase, and .about.9 .mu.g/ml T7
polymerase (Y639L/H784A/K378R) and 0.2 .mu.M template DNA. The RNA
was precipitated and loaded on 10% denaturing PAGE. The RNA
visualized by UV absorbance on the gel. The yields did not differ
greatly between the four leader sequences tested and are shown in
Table IA below (Relative transcription yields given):
TABLE-US-00014 TABLE 1B Pool Yield (relative to Pool 4) Pool 1 80%
Pool 2 88% Pool 3 130% Pool 4 100%
In particular embodiments, the above identified templates may be
used in the transcription methods and/or aptamer selection methods
of the invention.
Example 4D
MNA Transcription using P266L/Y639L/H784A/K378R Mutant T7 RNA
Polymerase
The following DNA template and primers were used to program a
polymerase chain reaction to generate a double-stranded
transcription template. N indicates a degenerate position with an
approximately equal probability of being each of ATGC, all
sequences are listed in the 5' to 3' direction:
TABLE-US-00015 PCR Template (ARC2118)
TAATACGACTCACTATAGGGGAGTACAATAACGT (SEQ ID NO 3)
TCTCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NGGATCGTTACGACTAGCATCGATG
5'-primer AAAAAAAAAAAAAAAAAAAAAAAAAAATAATACG (SEQ ID NO 115)
ACTCACTATAGGGGAGTACAATAACGTTCTCG 3'-primer CATCGATGCTAGTCGTAACG
(SEQ ID NO 116)
The resultant double-stranded transcription template was then used
to program 200 uL transcription mixtures for each sample as
follows: HEPES (200 mM), DTT (40 mM), Spermidine (2 mM), Triton
X-100 (0.01%), MgCl.sub.2 (8 mM), MnCl.sub.2 (2.5 mM), PEG-8000
(10% w/v), 1.5 mM each of 2'-OMe NTP, GMP 1 mM, 100-200 nM
transcription template, Inorganic Pyrophosphatase (1 unit), pH 7.5,
the T7 mutant polymerase P266L/Y639L/H784A/K378R was diluted as
indicated below. The transcription mixture was incubated at
37.degree. C. overnight (16 h).
After incubation, the mixtures were precipitated with isopropanol,
the resultant pellet was dissolved and quantitated using denaturing
PAGE (12.5% acrylamide) for 60 min at 25 W. The samples were
visualized and quantitated by UV shadow at 260 nm.
TABLE-US-00016 TABLE 2 Transcriptional Yield Normalized MNA Enzyme
Enzyme Concentration Transcript Yield K378R/Y639L/H784A 2.1
.mu.g/ml 100 P266L/K378R/Y639L/H784A 11 .mu.g/ml 130
P266L/K378R/Y639L/H784A 2.6 .mu.g/ml 65 P266L/K378R/Y639L/H784A
0.66 .mu.g/ml 13 P266L/K378R/Y639L/H784A 0.16 .mu.g/ml 8.3
Example 5
Aptamer Selection Using Y639L/H784A/K378R Mutant T7 RNA
Polymerase
A selection was performed to identify aptamers to human Ang2
(hereinafter "h-Ang2") using a pool consisting of 2'-OMe purine and
pyrimidine nucleotides (hereinafter "MNA"). The selection strategy
yielded high affinity aptamers to specific for h-Ang2.
Human Ang2 was purchased from R&D Systems, Inc. (Minneapolis,
Minn.). T7 RNA polymerase (Y639L/H784A/K378R) was expressed and
purified as described in Example 3 above. 2'-OMe purine and
pyrimidine nucleotides were purchased from TriLink BioTechnologies
(San Diego, Calif.).
Selection of Ang2 Aptamer
Pool Preparation
A DNA template with the sequence
5'-TAATACGACTCACTATAGGGGAGTACAATAACGTTCTCGNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG ARC2118 (SEQ ID NO 3) was
synthesized using an ABI EXPEDITE.TM. (Applied Biosystems, Foster
City, Calif.) DNA synthesizer, and deprotected by standard methods.
The templates were amplified with the primers
(5'-(GATCGATCGATCGATCGATCTAATACGACTCACTATAGGGGAGTACAATAACGTTC
TCG-3') (SEQ ID NO 118) and (5'-CATCGATGCTAGTCGTAACGATCC-3') (SEQ
ID NO 119) and then used as a template for in vitro transcription
with T7 RNA polymerase (Y639L/H784A/K378R). Transcriptions were
done using 200 mM Hepes, 40 mM DTT, 2 mM spermidine, 0.01% Triton
X-100, 10% PEG-8000, 8 mM MgCl.sub.2, 2.5 mM, MnCl.sub.2, 1.5 mM
mCTP, 1.5 mM mUTP, 1.5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01
units/.mu.L inorganic pyrophosphatase, and .about.9 .mu.g/mL T7
polymerase (Y639L/H784A/K378R) and 0.5 .mu.M template DNA to
generate the ARC2118 mRmY pool.
Selection
The selection was initiated by incubating of 330 pmoles
(2.times.10.sup.4 molecules) of MNA ARC 2118 pool with 100 pmoles
of protein in a final volume of 100 .mu.L selection buffer
(1.times. Dulbecco's PBS (DPBS)) for 1 hr at room temperature.
RNA-protein complexes and unbound RNA molecules were separated
using a 0.45 micron nitrocellulose spin column (Schleicher and
Schuell, Keene, N.H.). The column was pre-treated with KOH (Soak
column filter in 1 mL 0.5M KOH, 15 min RT; spin through. Soak
filter in 1 mL dH2O 5 min RT; spin through), washed 2.times.1 mL
1.times.PBS, and then the solution containing pool:Ang2 complexes
was added to the column and centrifuged at 1500.times.g for 2
minutes. The filter was washed twice with 500 .mu.L DPBS to remove
non-specific binders. RNA was eluted by addition of 2.times.100
.mu.L elution buffer (7 M urea, 100 mM sodium acetate, 3 mM EDTA,
pre-heated to 95.degree. C.) and then precipitated with ethanol.
The RNA was reverse transcribed with the ThermoScript RT-PCR.TM.
system (Invitrogen, Carlsbad, Calif.) according to the
manufacturer's instructions using the primer SEQ ID NO 119. The
cDNA was amplified by PCR with Taq polymerase (New England Biolabs,
Beverly, Mass.) according to the manufacturer's instructions using
SEQ ID NO 118 and SEQ ID NO 119. Templates were transcribed as
described above for pool preparation and purified on a denaturing
polyacrylamide gel.
Round 2 was performed with the same method as round 1. Rounds 3-12
were carried out with h-Ang2 immobilized on hydrophobic plates.
Each round of selection was initiated by immobilizing 20 pmoles of
h-Ang2 to the surface of a Nunc Maxisorp hydrophobic plate for 1
hour at room temperature in 100 .mu.L of 1.times.DPBS. The plate
was washed 5.times. with 120 .mu.L DPBS then incubated with
blocking buffer (1.times.DPBS, and 0.1 mg/mL BSA) for 1 hour. The
supernatant was then removed and the wells were washed 5 times with
120 .mu.L 1.times.DPBS. The pool RNA was incubated for 1 hour at
room temperature in empty wells then for 1 hour in a well that had
been previously blocked with 100 .mu.L blocking buffer. From round
3 forward, the target-immobilized wells were blocked for 1 hour at
room temperature in 100 .mu.L blocking buffer (1.times.PBS, 0.1
mg/mL tRNA, 0.1 mg/mL ssDNA and 0.1 mg/mL BSA) before the positive
selection step. In all cases, the pool RNA bound to immobilized
h-Ang2 was reverse transcribed directly in the selection plate by
the addition of reverse transcription ("RT") mix (3' primer, SEQ ID
NO 119, and Thermoscript RT, Invitrogen, Carlsbad, Calif.) followed
by incubation at 65.degree. C. for 1 hour. The resulting cDNA was
used as a template for PCR (Taq polymerase, New England Biolabs,
Mass.) and transcription as described for round 1. Conditions for
each round are in Table 3.
TABLE-US-00017 TABLE 3 Round Summary Round Pool (nM) Platform
Negative Buffer Competitor Target (nM) 1 3300 KOH None 1X None 1000
filter DPBS 2 1000 KOH KOH filter 1X None 1000 filter DPBS 3 1000
Plate Plate; BSA 1X None 200 plate DPBS 4 1000 Plate Plate; BSA 1X
None 200 plate DPBS 5 1000 Plate Plate; BSA 1X None 200 plate DPBS
6 1000 Plate Plate; BSA 1X None 200 plate DPBS 7 1000 Plate Plate;
BSA 1X None 200 plate DPBS 8 1000 Plate Plate; BSA 1X None 200
plate DPBS 9 1000 Plate Plate; BSA 1X 0.1 mg/mL 200 plate DPBS tRNA
10 1000 Plate Plate; BSA 1X 0.1 mg/mL 200 plate DPBS tRNA 11 1000
Plate Plate; BSA 1X 0.1 mg/mL 200 plate DPBS tRNA 12 1000 Plate
Plate; BSA 1X 0.1 mg/mL 200 plate DPBS tRNA
MNA Aptamer Binding Analysis
Dot blot binding assays were performed throughout the selections to
monitor the protein binding affinity of the pools. Trace
.sup.32P-endlabeled pool RNA was combined with h-Ang2 and incubated
at room temperature for 30 minutes in DPBS buffer in a final volume
of 30 .mu.L. The mixture was applied to a dot blot apparatus
(Minifold-1 Dot Blot, Acrylic, Schleicher and Schuell, Keene,
N.H.), assembled (from top to bottom) with nitrocellulose, nylon,
and gel blot membranes. RNA that is bound to protein is captured on
the nitrocellulose filter; whereas the non-protein bound RNA is
captured on the nylon filter. Enrichment for h-Ang2 binding was
seen starting at round 9. Round 9, 10 and 12 pool templates were
cloned using the TOPO TA cloning kit (Invitrogen, Carlsbad, Calif.)
according to the manufacturer's instructions and 26 unique clones
were chosen for chemical synthesis and dissociation constants
(K.sub.D) were determined. Briefly, the synthetic RNAs were 5'end
labeled with .gamma.-.sup.32P ATP and K.sub.D values were
determined using the dot blot assay and buffer conditions of
1.times.DPBS (w/Ca.sup.2+ and Mg.sup.2+) (Gibco, Catalog #14040,
Invitrogen, Carlsbad, Calif.). K.sub.DS were estimated fitting the
data to the equation: fraction RNA
bound=amplitude*(((AptConc+[h-Ang2]+K.sub.D)-SQRT((AptConc+[h-Ang2]+K.sub-
.D).sup.2-4(AptConc*[h-Ang2])))/(2*AptConc))+background. Results
are reported in Table 4 below.
Within the 26 unique sequences, 8 shared a similar motif and had
similar binding and inhibitory activity. These sequences are
identified as Family I. Family II comprises 2 sequences with a
shared motif that had similar binding and inhibitory
activities.
Analysis of MNA Aptamer Function
Elisa Assay
Some the aptamers were tested in an ELISA assay that was setup to
measure their ability to interfere with Ang2 binding to the Tie2
receptor. To capture Tie2 receptor, 150 ng of Tie2-Fc (R&D
systems 313-TI-100-CF, Minneapolis, Minn.) in 100 .mu.L of PBS (pH
7.4) was put onto a 96-well Maxisorb plate (NUNC #446612,
Rochester, N.Y.) and incubated overnight at 4.degree. C. During the
capture, 50 .mu.L of various concentrations of synthetic RNA were
mixed with 50 .mu.L of 3.6 nM Ang2 (200 ng/mL) (R&D systems,
623-AN-025/CF, Minneapolis, Minn.) (in PBS with 0.2% BSA) with
final Ang2 concentration at 1.8 nM (100 ng/mL) in PBS with 0.1% BSA
and incubated at room temperature for 1 hour. The capture solution
was removed after an overnight incubation and the plate was washed
with 200 .mu.L of TBST (25 mM Tris-HCl pH 7.5, 150 mM NaCl and
0.01% Tween 20) three times. The plate was then blocked with 200
.mu.L TBST containing 5% nonfat dry milk for 30 minutes at room
temperature. After blocking, the .mu.plate was washed with 200
.mu.L of TBST again three times at room temperature and synthetic
RNA:Ang2 mixture was added to the plate and incubated at room
temperature for 1 hours. The plate was then washed with 200 .mu.L
of TBST three times and 100 .mu.L of biotinylated goat anti-Ang2
antibody (1:1000; R&D Systems BAF623, Minneapolis, Minn.) was
added and incubated for 1 hour at room temperature. After three
washes with 200 .mu.L of TBST, 100 .mu.L of HRP linked Streptavidin
(1:200; R&D systems #DY998, Minneapolis, Minn.) was added and
incubated at room temperature for 0.5 hours. Then, the plate was
washed again with 200 .mu.L of TBST three times and 100 .mu.L of
TMP solution (Pierce, #34028) was added and incubated in the dark
at room temp for 5 minutes. A solution of 100 .mu.L containing 2 N
H.sub.2SO.sub.4 was added to stop the reaction and the plate was
read by SpectroMax at 450 nm. The results are given in the final
column of Table 4 below.
FACS Assay
Human umbilical vein endothelial cell ("HUVEC") (ATCC) and K293
cell, a cell line overexpressing human Tie2 receptor, were used to
determine the IC.sub.50 of specific MNA Ang2 aptamers that inhibit
binding of Ang2 to Tie2 receptor on the cell membrane. In brief,
recombinant mammalian expression vector pCDNA3.1-Tie2 was
transfected into 293 cells (ATCC, Manassas, Va.) and stable clones
were then obtained after selection with G418 (Invitrogen, Carlsbad,
Calif.). Flow cytometry demonstrated expression of Tie2 protein on
both HUVEC and K293 cells. An Ang2 titration assay further
determined the amounts of Ang2 (R&D Systems, Minneapolis,
Minn.) for aptamer inhibition assay on HUVEC and K293 cells which
were 1 and 0.1 .mu.g/mL, respectively.
In the flow cytometry binding assay, HUVEC and K293 cells
(2.times.10.sup.5 cells/well) were pelleted in V bottomed 96-well
plate and were subsequently resuspended and incubated in MNA
aptamer/Ang2 solutions for 2 hours. Aptamer/Ang2 solutions were
prepared by pre-incubation of different dosage of aptamers (100 nM,
33.3 nM, 11.1 nM, 3.7 nM, 1.2 nM, 0.411 nM, 0.137 nM, and 0.0456
nM) with Ang2 in FACs buffer (1% BSA, 0.2% sodium azide in PBS) for
30 min on ice. After three washes with FACs buffer, cells were
incubated 30 minutes with biotinylated anti-human Ang2 antibody (5
.mu.g/mL; R&D Systems, Minneapolis, Minn.), followed by another
30 minute incubation with Streptavidin PE (1:10; BD Biosciences,
San Jose, Calif.). FACS analysis was completed using FACScan (BD
Biosciences, San Jose, Calif.). The results are reported in Table 4
below.
TABLE-US-00018 TABLE 4 Summary of binding and functional results
for anti-Ang2 MNA aptamers IC.sub.50 (293-Tie2 IC.sub.50 MNA
Selection FACs) ELISA Aptamer Round Family K.sub.D (nM) (nM) (nM) 1
10 & 12 I 0.7 0.7 Not tested 2 10 & 12 I Not tested 0.5 Not
tested 3 12 I 0.2 0.5 Not tested 4 10 & 12 I 20.0 1.0 Not
tested 5 12 I 34.0 0.7 Not tested 6 10 & 12 I 9.0 0.5 1.0 7 12
I 17.0 0.5 0.3 8 10 II 19.0 1.6 1.5 9 10 I 120.0 Not tested Not
tested 10 12 I 70.0 Not tested Not tested 11 12 I No Not tested Not
tested binding 12 12 I 170.0 Not tested Not tested 13 12 I 82.0 Not
tested Not tested 14 12 I No Not tested Not tested binding 15 12 I
No Not tested Not tested binding 16 12 I No No Not tested binding
Inhibition 17 12 I 20.0 No Not tested Inhibition 18 12 I 90.0 Not
tested Not tested 19 12 II 25.0 1.1 2.4 20 12 I No No Not tested
binding Inhibition 21 12 I 2.9 0.5 Not tested 22 12 I 17.0 0.6 Not
tested 23 12 I No Not tested Not tested binding 24 12 I No Not
tested Not tested binding 25 12 I No No Not tested binding
Inhibition 26 12 I No Not tested Not tested binding
Example 6
Aptamer Selection Using Y639L/H784A/K378R Mutant T7 RNA
Polymerase
A selection was performed to identify aptamers to human IgE
(hereinafter "h-IgE") using a pool consisting of 2'-OMe purine and
pyrimidine nucleotides (hereinafter "mRmY"). The selection strategy
yielded high affinity aptamers specific for h-IgE.
Human IgE was purchased from Athens Research & Technology (Cat.
#16-16-090705 Athens, Ga.). T7 RNA polymerase (Y639L/H784A/K378R)
was expressed and purified as described in Example 3 above. 2'-OMe
purine and pyrimidine nucleotides were purchased from TriLink
BioTechnologies (San Diego, Calif.).
Selection of IgE Aptamer
Pool Preparation
A DNA template with the sequence
5'-TAATACGACTCACTATAGGGGAGTACAATAACGTTCTCGNNNNNNNNNNNNNNNNN
NNNNNNNNNNNNNGGATCGTTACGACTAGCATCGATG-3' ARC2118 (SEQ ID NO 3) was
synthesized using an ABI EXPEDITE.TM. (Applied Biosystems, Foster
City, Calif.) DNA synthesizer, and deprotected by standard methods.
The templates were amplified with the primers
(5'-(GATCGATCGATCGATCGATCTAATACGACTCACTATAGGGGAGTACAATAACGTTC
TCG-3') (SEQ ID NO 118) and (5'-CATCGATGCTAGTCGTAACGATCC-3') (SEQ
ID NO 119) and then used as a template for in vitro transcription
with T7 RNA polymerase (Y639L/H784A/K378R). Transcriptions were
done using 50 mM HEPES, 10 mM DTT, 0.5 mM spermidine, 0.0025%
Triton X-100, 10% PEG-8000, 8 mM MgCl.sub.2, 2.5 mM MnCl.sub.2, 1.5
mM mCTP, 1.5 mM mUTP, 1.5 mM mGTP, 1.5 mM mATP, 1 mM GMP, 0.01
units/.mu.L inorganic pyrophosphatase, and .about.9 .mu.g/mL mutant
T7 polymerase (Y639L/H784A/K378R) and 0.3 .mu.M template DNA to
generate the ARC2118 MNA pool
Selection
The selection was initiated by incubating of 330 pmoles
(2.times.10.sup.14 molecules) of MNA ARC 2118 pool with 24 pmoles
of protein bound to a BSA-blocked hydrophobic plate (Maxisorp
plate, Nunc, Rochester, N.Y.) in a final volume of 100 .mu.L
selection buffer (1.times. Dulbecco's PBS (DPBS) for 1 hr at room
temperature. The well was washed four times with 120 .mu.L DPBS to
remove non-specific binders. RNA was eluted and reverse transcribed
with the ThermoScript RT-PCR.TM. system (Invitrogen, Carlsbad,
Calif.) according to the manufacturer's instructions using the
primer SEQ ID NO 119. The cDNA was amplified by PCR with Taq
polymerase (New England Biolabs, Beverly, Mass.) according to the
manufacturer's instructions using SEQ ID NO 118 and SEQ ID NO 119.
Templates were transcribed as described above for pool preparation
and purified on a denaturing polyacrylamide gel.
All rounds were carried out with h-IgE immobilized on hydrophobic
plates. Each round of selection was initiated by immobilizing 24
pmoles of h-IgE to the surface of a Nunc Maxisorp hydrophobic plate
for 1 hour at room temperature in 100 .mu.L of 1.times.DPBS. The
plate was washed four times with 120 .mu.L DPBS then incubated with
blocking buffer (1.times.DPBS, and 0.1 mg/mL BSA) for 1 hour. The
supernatant was then removed and the wells were washed four times
with 120 .mu.L 1.times.DPBS. Starting at Round 2, the pool RNA was
incubated for 1 hour at room temperature in empty wells then for 1
hour in a well that had been previously blocked with 100 .mu.L
blocking buffer. From Round 2 forward, non-specific competitor was
added to the positive selection step (0.1 mg/mL tRNA, and 0.1 mg/mL
ssDNA). In all cases, the pool RNA bound to immobilized h-IgE was
reverse transcribed directly in the selection plate by the addition
of reverse transcription ("RT") mix (3' primer, SEQ ID NO 119, and
Thermoscript RT, Invitrogen, Carlsbad, Calif.) followed by
incubation at 65.degree. C. for 1 hour. The resulting cDNA was used
as a template for PCR (Taq polymerase, New England Biolabs,
Beverly, Mass.) and transcription as described for round 1.
Conditions for each round are in Table 5.
TABLE-US-00019 TABLE 5 Round Summary Round Pool (nM) Platform
Negative Buffer Competitor Washes Target 1 3300 Plate None 1X DPBS
None 4 .times. 120 .mu.L 24 pmols 2 500 Plate Plate; BSA plate 1X
DPBS 0.1 mg/mL tRNA; 4 .times. 120 .mu.L 24 pmols 0.1 mg/mL ssDNA 3
1000 Plate Plate; BSA plate 1X DPBS 0.1 mg/mL tRNA; 4 .times. 120
.mu.L 24 pmols 0.1 mg/mL ssDNA 4 1000 Plate Plate; BSA plate 1X
DPBS 0.1 mg/mL tRNA; 4 .times. 120 .mu.L 24 pmols 0.1 mg/mL ssDNA 5
1000 Plate Plate; BSA plate 1X DPBS 0.1 mg/mL tRNA; 4 .times. 120
.mu.L 24 pmols 0.1 mg/mL ssDNA 6 1000 Plate Plate; BSA plate 1X
DPBS 0.1 mg/mL tRNA; 4 .times. 120 .mu.L 24 pmols 0.1 mg/mL ssDNA 7
1000 Plate Plate; BSA plate 1X DPBS 0.1 mg/mL tRNA; 4 .times. 120
.mu.L 24 pmols 0.1 mg/mL ssDNA 8 1000 Plate Plate; BSA plate 1X
DPBS 0.1 mg/mL tRNA; 4 .times. 120 .mu.L 24 pmols 0.1 mg/mL ssDNA 9
1000 Plate Plate; BSA plate 1X DPBS 0.1 mg/mL tRNA; 4 .times. 120
.mu.L 24 pmols 0.1 mg/mL ssDNA 10 1000 Plate Plate; BSA plate 1X
DPBS 1.0 mg/mL tRNA; 8 .times. 120 .mu.L 24 pmols 1.0 mg/mL ssDNA
(last wash = 15 min.) 11 500 Plate Plate; BSA plate 1X DPBS 1.0
mg/mL tRNA; 8 .times. 120 .mu.L 24 pmols 1.0 mg/mL ssDNA (last wash
= 15 min.) 12 500 Plate Plate; BSA plate 1X DPBS 1.0 mg/mL tRNA; 8
.times. 120 .mu.L 24 pmols 1.0 mg/mL ssDNA (last wash = (15
min.)
MNA Aptamer Binding Analysis
Dot blot binding assays were performed throughout the selections to
monitor the protein binding affinity of the pools. Trace
32P-endlabeled pool RNA was combined with h-IgE and incubated at
room temperature for 30 minutes in DPBS buffer in a final volume of
30 .mu.L. The mixture was applied to a dot blot apparatus
(Minifold-1 Dot Blot, Acrylic, Schleicher and Schuell, Keene,
N.H.), assembled (from top to bottom) with nitrocellulose, nylon,
and gel blot membranes. RNA that is bound to protein is captured on
the nitrocellulose filter; whereas the non-protein bound RNA is
captured on the nylon filter. Enrichment for h-IgE binding was seen
starting at Round 8. Round 5, 8 and 12 pool templates were cloned
using the TOPO TA cloning kit (Invitrogen, Carlsbad, Calif.)
according to the manufacturer's instructions. The sequencing data
revealed that the Round 8 pool had converged on a single major
clone that comprised 59% of the total sequences. This major clone
and three possible minimers were chosen for chemical synthesis and
dissociation constants (KD) were determined. Briefly, the synthetic
RNAs were 5'end labeled with .gamma.-.sup.32P ATP and KD values
were determined using the dot blot assay and buffer conditions of
1.times.DPBS (w/Ca2+ and Mg2+) (Gibco, Catalog #14040, Invitrogen,
Carlsbad, Calif.). KDs were estimated fitting the data to the
equation: fraction RNA
bound=amplitude*(((AptConc+[h-IgE]+KD)-SQRT((AptConc+[h-IgE]+KD)2-4(AptCo-
nc*[h-IgE])))/(2*AptConc))+background. The major clone had a
K.sub.D of about 800 pM. The best binding minimer, was also tested
for binding to monkey IgE (m-IgE), but did not demonstrate
cross-reactive binding to the monkey IgE protein. This lack of
cross-reactivity for was also confirmed by ELISA. Minimers with an
inverted dT on the 3' end, was used as the parent molecule for the
medicinal chemistry process.
Medicinal Chemistry
The chemical composition of one of the IgE specific MNA minimers
(FIG. 11) was altered to improve affinity, and potency while
maintaining plasma stability of the compound. The process included
the design, synthesis and evaluation of a series of derivatives of
the minimized IgE aptamer where each derivative of the series
comprised a single modification at each occurrence of a
predetermined nucleotide to determine which residues tolerated
substitution. The first set of modifications was the substitution
of a deoxy nucleotide for each unique 2'-OMe nucleotide. In a
separate round of modification, a series of derivatives was
synthesized in which each derivative comprised a single
phosphorothioate modification at a different internucleotide
linkage position. Data generated in these initial phases of
modification were used to establish a structure activity
relationship (SAR) for the minimized aptamer. In a subsequent phase
of modification, aptamers were synthesized and tested with
composite sets of substitutions that were designed based on the
initial SAR data. From the panel of composite substitutions, an
aptamer 39 nucleotides in length with two 2'-OMe to 2'-deoxy
substitutions introduced into its composition, was identified. In
addition, a resulting modified minimized aptamer, 39 nucleotides in
length with one 2'-OMe to 2'-deoxy substitution and four phosphate
to phosphorothioate substitutions incorporated into its
composition, was identified. As shown in FIG. 12, this
deoxy/phosphorothioate modified aptamer, demonstrates increased
binding affinity compared to both the minimized but unmodified
parent aptamer as well as the parent minimized aptamer having two
deoxy for 2'-OMe substitutions.
Serum Stability
The minimized unmodified parent and the deoxy/phosphorothioate
modified aptamer were assayed to determine their stability in
human, rat and monkey serums. Each aptamer was added to 1 ml of
pooled serum to a final concentration of 5 .mu.M in 90% serum. The
aptamers were incubated at 37.degree. C. with shaking and time
points were taken at 0, 0.5, 1, 4, 24, 48, 72, and 98 hours. At
each time point, 90 .mu.l of stock from the incubated samples was
added to 10 .mu.l of 0.5M EDTA and frozen at -20.degree. C. for
later stability analysis using a BIACORE 2000 system.
All biosensor binding measurements were performed at 25.degree. C.
using a BIACORE 2000 equipped with a research-grade CM5 biosensor
chip (BIACORE Inc., Piscataway, N.J.). Purified recombinant human
IgE (Athens Research & Technology, Athens, Ga.) was immobilized
to the biosensor surface using amino-coupling chemistry. To achieve
this, the surfaces of two flow cells were first activated for 7 min
with a 1:1 mixture of 0.1 M NHS (Nhydroxysuccinimide) and 0.4 M EDC
(3-(N,Ndimethylamine) propyl-N-ethylcarbodiimide) at a flow rate of
5 .mu.l/min. After surface activation, one flow cell was injected
with 50 .mu.g/ml of IgE at 10 .mu.l/min for 20 min to allow for
establishment of covalent bonds to the activated surface. Next, 1 M
ethanolamine hydrochloride pH 8.5 was injected for 7 min at 5
.mu.l/min to inactivate residual esters. For flow cell used as
blank, 1 M ethanolamine hydrochloride pH 8.5 was injected for 7 min
to inactivate residual esters without protein injection.
A set of aptamer standards was run through the prepared chip to
generate a standard curve before all the time-points were analyzed.
To establish a standard curve, aptamers were serially diluted (from
200 nM to 12.5 nM) into HBS-P buffer (10 mM HEPES pH 7.4, 150 mM
NaCl, 0.005% Surfactant 20) supplemented with 4% human serum and 50
mM EDTA. All diluted samples were injected into Biacore 2000 for
binding at 20 .mu.l/min for 5 min and wait for 3 minutes. To
regenerate the chip, 1N NaCl was injected for 60 seconds at 30
.mu.l/min. RU peak response at the end of binding phase was plotted
against aptamer concentration and a standard curve was generated
using a Four-Parameter logistic function. To measure the active
aptamer concentration in human, rat, and monkey serums, time-point
samples were diluted 22.5-fold in HBS-P to make the final serum
concentration at 4% immediately prior to injection into the Biacore
2000. Functional aptamer concentrations at each serum incubation
period were calculated by converting from RU response unit to
concentration using standard curve generated above. As an
additional quality control measure, two aptamer standards were
independently tested at the end of experiment to make sure the
BIACORE-measured concentrations are less than 20% deviated from
standards. The minimized unmodified parent and the
deoxy/phosphorothioate modified aptamer were both determined to be
greater than 90% active at 98 hours in human, rat, and monkey
serums.
The invention having now been described by way of written
description and example, those of skill in the art will recognize
that the invention can be practiced in a variety of embodiments and
that the description and examples above are for purposes of
illustration and not limitation of the following claims.
SEQUENCE LISTINGS
1
1511883PRTArtificial Sequencechemically synthesized modified T7
polymerase Y639/H784A 1Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe
Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp
His Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu Gln Leu Ala Leu Glu His
Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg Phe Arg Lys Met Phe Glu
Arg Gln Leu Lys Ala Gly Glu Val 50 55 60Ala Asp Asn Ala Ala Ala Lys
Pro Leu Ile Thr Thr Leu Leu Pro Lys65 70 75 80Met Ile Ala Arg Ile
Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95Gly Lys Arg Pro
Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110Ala Val
Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120
125Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu
Ala Lys145 150 155 160His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn
Lys Arg Val Gly His 165 170 175Val Tyr Lys Lys Ala Phe Met Gln Val
Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu
Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205Ser Ile His Val Gly
Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220Gly Met Val
Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235
240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro
Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly
Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro Leu Ala Leu Val Arg
Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg Tyr Glu Asp Val Tyr
Met Pro Glu Val Tyr Lys Ala Ile305 310 315 320Asn Ile Ala Gln Asn
Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335Val Ala Asn
Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350Pro
Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360
365Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu
Glu Phe385 390 395 400Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His
Lys Ala Ile Trp Phe 405 410 415Pro Tyr Asn Met Asp Trp Arg Gly Arg
Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met
Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445Gly Lys Pro Ile Gly
Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460Ala Asn Cys
Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475
480Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys
Phe Leu 500 505 510Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His
Gly Leu Ser Tyr 515 520 525Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly
Ser Cys Ser Gly Ile Gln 530 535 540His Phe Ser Ala Met Leu Arg Asp
Glu Val Gly Gly Arg Ala Val Asn545 550 555 560Leu Leu Pro Ser Glu
Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575Lys Val Asn
Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590Glu
Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600
605Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala
Leu Gly625 630 635 640Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu
Glu Asp Thr Ile Gln 645 650 655Pro Ala Ile Asp Ser Gly Lys Gly Leu
Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys
Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685Val Val Ala Ala Val
Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700Leu Leu Ala
Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715
720Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met
Phe Leu 740 745 750Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn
Lys Asp Ser Glu 755 760 765Ile Asp Ala His Lys Gln Glu Ser Gly Ile
Ala Pro Asn Phe Val Ala 770 775 780Ser Gln Asp Gly Ser His Leu Arg
Lys Thr Val Val Trp Ala His Glu785 790 795 800Lys Tyr Gly Ile Glu
Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815Ile Pro Ala
Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830Val
Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840
845Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser
Asp Phe865 870 875 880Ala Phe Ala2883PRTArtificial
Sequencechemically synthesized modified T7 polymerase
Y639L/H784A/K378R 2Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe Ser
Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp His
Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu Gln Leu Ala Leu Glu His Glu
Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg Phe Arg Lys Met Phe Glu Arg
Gln Leu Lys Ala Gly Glu Val 50 55 60Ala Asp Asn Ala Ala Ala Lys Pro
Leu Ile Thr Thr Leu Leu Pro Lys65 70 75 80Met Ile Ala Arg Ile Asn
Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95Gly Lys Arg Pro Thr
Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110Ala Val Ala
Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120 125Ala
Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala 130 135
140Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu Ala
Lys145 150 155 160His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn Lys
Arg Val Gly His 165 170 175Val Tyr Lys Lys Ala Phe Met Gln Val Val
Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu Ala
Trp Ser Ser Trp His Lys Glu Asp 195 200 205Ser Ile His Val Gly Val
Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220Gly Met Val Ser
Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235 240Ser
Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr 245 250
255Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro Cys Val
260 265 270Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly Gly Tyr
Trp Ala 275 280 285Asn Gly Arg Arg Pro Leu Ala Leu Val Arg Thr His
Ser Lys Lys Ala 290 295 300Leu Met Arg Tyr Glu Asp Val Tyr Met Pro
Glu Val Tyr Lys Ala Ile305 310 315 320Asn Ile Ala Gln Asn Thr Ala
Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335Val Ala Asn Val Ile
Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350Pro Ala Ile
Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360 365Met
Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val 370 375
380Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu
Phe385 390 395 400Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys
Ala Ile Trp Phe 405 410 415Pro Tyr Asn Met Asp Trp Arg Gly Arg Val
Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met Thr
Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445Gly Lys Pro Ile Gly Lys
Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460Ala Asn Cys Ala
Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475 480Phe
Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490
495Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu
500 505 510Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His Gly Leu
Ser Tyr 515 520 525Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys
Ser Gly Ile Gln 530 535 540His Phe Ser Ala Met Leu Arg Asp Glu Val
Gly Gly Arg Ala Val Asn545 550 555 560Leu Leu Pro Ser Glu Thr Val
Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575Lys Val Asn Glu Ile
Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590Glu Val Val
Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605Val
Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615
620Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu
Gly625 630 635 640Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu
Asp Thr Ile Gln 645 650 655Pro Ala Ile Asp Ser Gly Lys Gly Leu Met
Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys Leu
Ile Trp Glu Ser Val Ser Val Thr 675 680 685Val Val Ala Ala Val Glu
Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700Leu Leu Ala Ala
Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715 720Lys
Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp 725 730
735Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu
740 745 750Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp
Ser Glu 755 760 765Ile Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro
Asn Phe Val Ala 770 775 780Ser Gln Asp Gly Ser His Leu Arg Lys Thr
Val Val Trp Ala His Glu785 790 795 800Lys Tyr Gly Ile Glu Ser Phe
Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815Ile Pro Ala Asp Ala
Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830Val Asp Thr
Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845Phe
Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855
860Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp
Phe865 870 875 880Ala Phe Ala393DNAArtificial Sequencechemically
synthesized DNA transcription template ARC 2118 3taatacgact
cactataggg gagtacaata acgttctcgn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnng
gatcgttacg actagcatcg atg 93493DNAArtificial Sequencechemically
synthesized DNA transcription template ARC 2119 4taatacgact
cactataggg ggtgatattg acgttctcgn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnng
gatcgttacg actagcatcg atg 93593DNAArtificial Sequencechemically
synthesized DNA transcription template ARC 2120 5taatacgact
cactataggg gtgcgcggtt acgttctcgn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnng
gatcgttacg actagcatcg atg 93693DNAArtificial Sequencechemically
synthesized DNA transcription template ARC 2121 6taatacgact
cactataggg ggagggggtg ccgttctcgn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnng
gatcgttacg actagcatcg atg 93781DNAArtificial Sequencechemically
synthesized DNA transcription template ARC1140 7taatacgact
cactataggg gnnnnnnnnn nnnnnnnnnn nacgtaaccg gttaaacccg 60ggtcgatgca
gtaagctagc t 81817DNAArtificial Sequencechemically synthesized
5'-phosphorylated T7 promoter oligonucleotide 8tatagtgagt cgtatta
17921DNAArtificial Sequencechemically synthesized ligation splint
9taatacgact cactataggg g 211082DNAArtificial Sequencechemically
synthesized sequence 10taatacgact cactataggg ggtggggcca atggcgggat
atacgtaacc ggttataccc 60gggtcgatgc agtaagctag ct
821180DNAArtificial Sequencechemically synthesized sequence
11taatacgact cactataggg gatgtacata tgtattcgtg acgtgaccgg ttaaacccgg
60gtcgatgcag taagctagct 801282DNAArtificial Sequencechemically
synthesized sequence 12taatacgact cactataggg ggagcgggga gacgtagtca
tcacgtagcc ggttaaaccc 60gggtcgatgc agtaagctag ct
821379DNAArtificial Sequencechemically synthesized sequence
13taatacnact cactataggg ggtgggggtg gtggtgataa cgtaaccggt taaacccggg
60tcgatgcagt aagctagct 791483DNAArtificial Sequencechemically
synthesized sequence 14taatacgact cactataggg gggtgtcacc agatatgcct
tgaacgtaac ccgttaaacc 60cgggtcgatg cagtaagcta gct
831582DNAArtificial Sequencechemically synthesized sequence
15taatacgact cactataggg ggtagggggc acgcactaac caacgtaacc ggttaaaccc
60gggtcgatgc agtaagctag ct 821643DNAArtificial Sequencechemically
synthesized sequence 16taatacgact cactataggg ggagggggtg ctgaccncaa
aca 431781DNAArtificial Sequencechemically synthesized sequence
17taatacgact cactataggg gtggggctcg gatgagacaa tacgtaaccg gttaaacccg
60ggtcgatgca gtaagctagc t 811883DNAArtificial Sequencechemically
synthesized sequence 18taatacgact cactataggg gggggtgggt aggcgagcac
tccacgtaac cagttaaacc 60cgggtcgatg cagtaagcta gct
831982DNAArtificial Sequencechemically synthesized sequence
19taatacgact cactataggg gggaaggacg agcagacgag caacgtaacc tgttaaaccc
60gggtcgatgc agtaagctag ct 822085DNAArtificial Sequencechemically
synthesized sequence 20taatacgact cactataggg gggggcggtt agagtgtaag
taccgacgta accggttaaa 60cccgggtcga tgcagtaagc tagct
852179DNAArtificial Sequencechemically synthesized sequence
21taatacgact cactataggg gggttgctgt tagtaacgcc acgtaaccgg ttaaacttgg
60tcgatgcagt aagctagct 792284DNAArtificial Sequencechemically
synthesized sequence 22taatacgact cactataggg gggcgggaga atgttatata
gttacggtaa ccggttaaac 60ccgggtcgat gcagtaagct agct
842381DNAArtificial Sequencechemically synthesized sequence
23taatacgact cactataggg gaaaggggcg gtatggtaca cacgtaacag gttaaacccg
60ggtcgatgca gtaagctagc t 812480DNAArtificial Sequencechemically
synthesized sequence 24taatacgact cactataggg gggacgtgtt agcattccag
aattcgtaac ctaaacccgg 60gtcgatgcag taagctagct 802581DNAArtificial
Sequencechemically synthesized sequence 25taatacgact cactataggg
ggcgtgggag ataggttcaa ggacgtaccg gttatacccg 60ggtcgatgca gtaagctagc
t 812683DNAArtificial Sequencechemically synthesized sequence
26taatacgact cactataggg
gggctccgtg ctatcgtcgg ataacgtaac ccgttaaacc 60cgggtcgatg cagtaagcta
gct 832783DNAArtificial Sequencechemically synthesized sequence
27taatacgact cactataggg ggggagaagg tcttaaggtc gccaacgtaa ctgttaaacc
60cgggtcgatg cagtaagcta gct 832883DNAArtificial Sequencechemically
synthesized sequence 28taatacgact cactataggg ggggcatacg agtttaggtg
gagacgtaac cggttaaacc 60cgggtcgatg cagtaagcta gct
832982DNAArtificial Sequencechemically synthesized sequence
29taatacgact cactataggg ggatgatgac ttccgcgtta atacgttacc ggttaaaccc
60gggtcgatgc agtaagctag ct 823075DNAArtificial Sequencechemically
synthesized sequence 30taatacgact cactataggg gtgggacgcc gtctgagtat
aacgtacccg gtcgggtcga 60tgcagtaagc tagct 753187DNAArtificial
Sequencechemically synthesized sequence 31taatacgact cactataggg
ggggggggac gtaatcggct atcgttcacg taaccggtta 60aacccgggtc gatgcagtaa
agggcga 873284DNAArtificial Sequencechemically synthesized sequence
32taatacgact cactataggg tgggacgggc agcgtggatg taggacgtaa ccggttaaac
60gcgggtcgat gcagtaagct agct 843383DNAArtificial Sequencechemically
synthesized sequence 33taatacgact cactataggg gggtttgtct gaagtgaagc
agaacgtaac cggttaatcc 60cgggtcgatg cagtaagcta gct
833484DNAArtificial Sequencechemically synthesized sequence
34taatacgact cactataggg ggggagggca catcatcgta tcaaacgtaa ccagttaatc
60ccgggtcgat gcagtaagct agct 843581DNAArtificial Sequencechemically
synthesized sequence 35taatacgact cactataggg gaggctagag gacgcgacag
aacgtaaccg gttaaacccg 60ggtcgatgca gtaagctagc t 813681DNAArtificial
Sequencechemically synthesized sequence 36taatacgact cactataggg
ggcgatcgcg aagggatttc aacgtaaccg gttaaacccg 60ggtcgatgca gtaagctagc
t 813783DNAArtificial Sequencechemically synthesized sequence
37taatacgact cactataggg gggtagggaa agattacggg gctacgtaac cggttatacc
60tgggtcgatg cagtaagcta gct 833875DNAArtificial Sequencechemically
synthesized sequence 38taatacgact cactataggg gtggctatgg ctaacacgta
accggttata cccgggtcga 60tgcagtaagc tagct 753986DNAArtificial
Sequencechemically synthesized sequence 39taatacgact cactataggg
ggggggcggt ggctgtgcaa gcggaaacgt aaccggttaa 60acccgggtcg atgcagtaag
ctagct 864083DNAArtificial Sequencechemically synthesized sequence
40taatacgact cactataggg gggtgggggc acggtactga gttacgttac cggttaaacc
60cgggtcgatg cagtaagcta gct 834178DNAArtificial Sequencechemically
synthesized sequence 41taatacgact cactataggg gggagtgggg acaattagaa
gatgacgtaa ccgtccgggt 60cgatgcagta agctagct 784281DNAArtificial
Sequencechemically synthesized sequence 42taatacnact cactataggg
gtgcagtgag gagcgacnag tacgttaccg gttaaatccg 60agtcgatgca gtaagctagc
t 814383DNAArtificial Sequencechemically synthesized sequence
43taatacnact cactataggg ggacgggcac tgtggatgat ttaacgttac cggttaaacc
60cgagtcgatg cagtaagcta gct 834440DNAArtificial Sequencechemically
synthesized sequence 44taatacnact cactataggg gtcgatgcag taagctagct
404581DNAArtificial Sequencechemically synthesized sequence
45taatacnact cactataggg ggtgatattg acctctaaca gcacgtaacc ggttaaaccc
60ggtcgatgca gtaagctagc t 814679DNAArtificial Sequencechemically
synthesized sequence 46taatacgact cactataggg gggggggtgc agaggatgca
tccaagctcg taatcggtgg 60tcgatgcagt aagctagct 794784DNAArtificial
Sequencechemically synthesized sequence 47taatacgact cactataggg
gggggcgggt gcttgtgcct aatcacgtaa ccggttaaac 60ccgggtcgat gcagtaagct
agct 844879DNAArtificial Sequencechemically synthesized sequence
48taatacgact cactataggg gtttggtaat cgaacgtgga acgcaaccgg tttaaccggg
60tcgatgcagt aagctagct 794982DNAArtificial Sequencechemically
synthesized sequence 49taatacgact cactataggg gggatggaag aggcttgata
tcacgtaacc ggttaaacct 60gggtcgatgc agtaagctag ct
825076DNAArtificial Sequencechemically synthesized sequence
50taatacgact cactataggg ggttatacta actctgtaca caacgtaacc ggccgggtcg
60atgcagtaag ctagct 765181DNAArtificial Sequencechemically
synthesized sequence 51taatacgact cactataggg gtataggggg ggtatcggtg
tacgtaaccg gttaaacccg 60ggtcgatgca gtaagctagc t 815281DNAArtificial
Sequencechemically synthesized sequence 52taatacgact cactataggg
gagtacaata aggttccgag aacgcgaccg gttaaacccg 60ggtcgatgca gtaagctagc
t 815382DNAArtificial Sequencechemically synthesized sequence
53taatacgact cactataggg gtgcgcggtt acaaggcaac atacgtaacc ggttaaaccc
60gggtcgatgc agtaagctag ct 825483DNAArtificial Sequencechemically
synthesized sequence 54taatacnact cactataggg gggacggggt gacaaagtgt
cnaacgtaac cggttaaacc 60cgggtcgatg cagtaagcta gct
835581DNAArtificial Sequencechemically synthesized sequence
55taatacgact cactataggg gagacggcgg tacaagtcca tatgtaaccg gttaaacccg
60ggtcgatgca gtaagctagc t 815681DNAArtificial Sequencechemically
synthesized sequence 56taatacgact cactataggg gagtgggggc ttctcgttgc
cacgtaaccg cttaaacccg 60ggtcgatgca gtaagctagc t 815783DNAArtificial
Sequencechemically synthesized sequence 57taatacgact cactataggg
gggctgagcg tgtttgaggg accacgttac cggttaaacc 60cgggtcgatg cagtaagcta
gct 835883DNAArtificial Sequencechemically synthesized sequence
58taatacgact cactataggg gggtgggcgc aatgaaaagt tgggcgtaac cggttcaacc
60cgggtcgatg cagtaagcta gct 835981DNAArtificial Sequencechemically
synthesized sequence 59taatacgact cactataggg ggtagtgaag taaggcagtg
ttacgtaacc ggtgaacccg 60ggtcgatgca gtaagctagc t 816083DNAArtificial
Sequencechemically synthesized sequence 60taatacgact cactataggg
gggagggtgg gctagaacac acaacgtaac cggttaaacc 60cgggtcgatg cagtaagcta
gct 836184DNAArtificial Sequencechemically synthesized sequence
61taatacgact cactataggg ggggagagag gcggttacgt agggacgtta ccgattgaac
60tcaggtcgat gcagtaagct agct 846287DNAArtificial Sequencechemically
synthesized sequence 62taatacgact cactataggg ggggggggcg aataggtagg
gcgacgaacg ttaccggtta 60aacccgggtc gatgcagtaa gctagct
876382DNAArtificial Sequencechemically synthesized sequence
63taatacgact cactataggg ggagaggagg tccggctaga caacgtaacc ggttaaaccc
60gggtcgatgc agtaagctag ct 826469DNAArtificial Sequencechemically
synthesized sequence 64taatacgact cactataggg gggaggacgg gtcgtactgt
taaacctggg tcgatgcagt 60aagctagct 696581DNAArtificial
Sequencechemically synthesized sequence 65taatacgact cactataggg
ggcgcaacaa cgggaagtat acgtaaccgg tttaaacccg 60ggtcgatgca gtaagctagc
t 816682DNAArtificial Sequencechemically synthesized sequence
66taatacgact cactataggg ggaaggaaca cgcacatgca taacgtaact ggttgacccc
60gggtcgatgc agtaagctag ct 826781DNAArtificial Sequencechemically
synthesized sequence 67taatacgact cactataggg gagtggggag tactgtggac
aacgtgaccg gttaaacccg 60ggtcgatgca gtaagctagc t 816840DNAArtificial
Sequencechemically synthesized sequence 68taatacgact cactataggg
gtcgatgcag taagctagct 406985DNAArtificial Sequencechemically
synthesized sequence 69taatacgact cactataggg gggggggcta gggcggtcgg
atcggacgta accagttaaa 60cccgggtcga tgcagtaagc tagct
857083DNAArtificial Sequencechemically synthesized sequence
70taatacgact cactataggg ggggtggggg ttgctacatg ccctcgtaac cggttaagcc
60caggtcgatg cagtaagcta gct 837183DNAArtificial Sequencechemically
synthesized sequence 71taatacgact cactataggg gggtggcgac gatggagaga
ataacgtaat cggttaaacc 60cgggtcgatg cagtaagcta gct
837281DNAArtificial Sequencechemically synthesized sequence
72taatacgact cactataggg ggtaggcggg cctcatcaac aacgcaaccg gttaaacccg
60ggtcgatgca gtaagctagc t 817382DNAArtificial Sequencechemically
synthesized sequence 73taatacgact cactataggg ggtggctggt aaggacacaa
acacgtaact cgttaaaccc 60gggtcgatgc agtaagctag ct
827483DNAArtificial Sequencechemically synthesized sequence
74taatacgact cactataggg gggcgggcag cgcttataga tccacgtaac cggttaaacc
60cgggtcgatg cagtaagcta gct 837586DNAArtificial Sequencechemically
synthesized sequence 75taatacgact cactataggg ggggggtatc tgcggttagg
ctatcgacgt acccagttaa 60acccgggtcg atgcagtaag ctagct
867682DNAArtificial Sequencechemically synthesized sequence
76taatacgact cactataggg ggggtagggg acatcatagg tatacgtaac cggttaaccc
60gggtcgatgc agtaagctag ct 827781DNAArtificial Sequencechemically
synthesized sequence 77taatacgact cactataggg gcgcgtgcgt gtatccatta
aacgtgactg gttaaacccg 60ggtcgatgca gtaagctagc t 817884DNAArtificial
Sequencechemically synthesized sequence 78taatacgact cactataggg
gggggagcgt ggatcttgag tgtatacgta accggttaaa 60cccggtcgat gcagtaagct
agct 847981DNAArtificial Sequencechemically synthesized sequence
79taatacgact cactataggg gatggagagg agtgtacgca tatacaaccg gttaaacccg
60ggtcgatgca gtaagctagc t 818080DNAArtificial Sequencechemically
synthesized sequence 80taatacgact cactataggg gcgggtggtc gcgatggtta
acgtaactgg ttaaacccgg 60gtcgatgcag taagctagct 808190DNAArtificial
Sequencechemically synthesized sequence 81taatacgact cactataggg
gggggggggg acgttagctt ctctgtattt acgtaaccgg 60ttaagcccgg gtcgatgcag
taagctagct 908240DNAArtificial Sequencechemically synthesized
sequence 82taatacgact cactataggg gtcgatgcag taagctagct
408383DNAArtificial Sequencechemically synthesized sequence
83taatacgact cactataggg gggatggagt gggtgcaaat aanacgtaac tggttaaacc
60cgggtcgatg cagtaagcta gct 838481DNAArtificial Sequencechemically
synthesized sequence 84taatacgact cactataggg gagngtgagg ggtgaatant
aangtaancn gttaaacctg 60ggtcgatgnn ntannctngn t 818540DNAArtificial
Sequencechemically synthesized sequence 85naatnngact cacaanaggg
gtcgatgcag taagctagct 408684DNAArtificial Sequencechemically
synthesized sequence 86taatacgact cactataggg gggggtgacg tacggatcta
agtaacgtaa ccggttaaac 60ccgggtcgat gcagtaagct agct
848780DNAArtificial Sequencechemically synthesized sequence
87taatacgact cactataggg gagggacaga cactttgtag acgtaaccag ttaaacccgg
60gtcgatgcag taagctagct 808883DNAArtificial Sequencechemically
synthesized sequence 88taatacgact cactataggg gggggacttg gcactacgta
acaacgtaac cgcttaaacc 60cgggtcgatg cagtaagcta gct
838986DNAArtificial Sequencechemically synthesized sequence
89taatacgact cactataggg gggggggcct ctcgaccaaa agcccaacgt aaccggttaa
60acccgggtcg atgcagtaag ctagct 869085DNAArtificial
Sequencechemically synthesized sequence 90taatacnact cactataggg
ggggggggat agtcatgact gataaaacgt aactgttgag 60cccgggtcga tgcagtaagc
tagct 859181DNAArtificial Sequencechemically synthesized sequence
91taatacgact cactataggg gacagtgcta gtggaatagc aacgtaacca gttaaacccg
60ggtcgatgca gtaagctagc t 819281DNAArtificial Sequencechemically
synthesized sequence 92taatacgact cactataggg gacgaccact atactccgag
aacgtaaccg gttaaacccg 60ggtcgatgca gtaagctagc t 819382DNAArtificial
Sequencechemically synthesized sequence 93taatacgact cactataggg
ggatggaggc gtagtgtagt caacgttacc ggttaaaccc 60gggtcgatgc agtaagctag
ct 829482DNAArtificial Sequencechemically synthesized sequence
94taatacgact cactataggg gggaggtata gatggaatgg ttatgtaacc tgttaaaccc
60gggtcgatgc agtaagctag ct 829581DNAArtificial Sequencechemically
synthesized sequence 95taatacgact cactataggg gtggggagga ccacttagat
aacgtcaccg gttaaacccg 60ggtcgatgca gtaagctagc t 819683DNAArtificial
Sequencechemically synthesized sequence 96taatacgact cactataggg
gggatagggg cgagagagtc acaacgtaac cggttaatcc 60cgggtcgatg cagtaagcta
gct 839783DNAArtificial Sequencechemically synthesized sequence
97taatacgact cactataggg gggggatggc cgaatcataa aataacgtaa ccgttagacc
60cgggtcgatg cagtaagcta gct 839878DNAArtificial Sequencechemically
synthesized sequence 98taatacgact cactataggg ggcgattgct gagtcagttc
gtaatcggtt aaacccgggt 60cgatgcagta agctagct 789985DNAArtificial
Sequencechemically synthesized sequence 99taatacgact cactataggg
gggggaggat ccgaaacaca gggatccgta accggttaaa 60gccgggtcga tgcagtaagc
tagct 85100883PRTArtificial Sequencechemically synthesized modified
T7 polymerase Y639L 100Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe
Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp
His Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu Gln Leu Ala Leu Glu His
Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg Phe Arg Lys Met Phe Glu
Arg Gln Leu Lys Ala Gly Glu Val 50 55 60Ala Asp Asn Ala Ala Ala Lys
Pro Leu Ile Thr Thr Leu Leu Pro Lys65 70 75 80Met Ile Ala Arg Ile
Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95Gly Lys Arg Pro
Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110Ala Val
Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120
125Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu
Ala Lys145 150 155 160His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn
Lys Arg Val Gly His 165 170 175Val Tyr Lys Lys Ala Phe Met Gln Val
Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu
Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205Ser Ile His Val Gly
Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220Gly Met Val
Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235
240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln Pro
Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly
Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro Leu Ala Leu Val Arg
Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg Tyr Glu Asp Val Tyr
Met Pro Glu Val Tyr Lys Ala Ile305 310 315 320Asn Ile Ala Gln Asn
Thr Ala Trp Lys Ile Asn Lys
Lys Val Leu Ala 325 330 335Val Ala Asn Val Ile Thr Lys Trp Lys His
Cys Pro Val Glu Asp Ile 340 345 350Pro Ala Ile Glu Arg Glu Glu Leu
Pro Met Lys Pro Glu Asp Ile Asp 355 360 365Met Asn Pro Glu Ala Leu
Thr Ala Trp Lys Arg Ala Ala Ala Ala Val 370 375 380Tyr Arg Lys Asp
Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu Glu Phe385 390 395 400Met
Leu Glu Gln Ala Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410
415Pro Tyr Asn Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe
420 425 430Asn Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu
Ala Lys 435 440 445Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu
Lys Ile His Gly 450 455 460Ala Asn Cys Ala Gly Val Asp Lys Val Pro
Phe Pro Glu Arg Ile Lys465 470 475 480Phe Ile Glu Glu Asn His Glu
Asn Ile Met Ala Cys Ala Lys Ser Pro 485 490 495Leu Glu Asn Thr Trp
Trp Ala Glu Gln Asp Ser Pro Phe Cys Phe Leu 500 505 510Ala Phe Cys
Phe Glu Tyr Ala Gly Val Gln His His Gly Leu Ser Tyr 515 520 525Asn
Cys Ser Leu Pro Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535
540His Phe Ser Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val
Asn545 550 555 560Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly
Ile Val Ala Lys 565 570 575Lys Val Asn Glu Ile Leu Gln Ala Asp Ala
Ile Asn Gly Thr Asp Asn 580 585 590Glu Val Val Thr Val Thr Asp Glu
Asn Thr Gly Glu Ile Ser Glu Lys 595 600 605Val Lys Leu Gly Thr Lys
Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly 610 615 620Val Thr Arg Ser
Val Thr Lys Arg Ser Val Met Thr Leu Ala Leu Gly625 630 635 640Ser
Lys Glu Phe Gly Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650
655Pro Ala Ile Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln
660 665 670Ala Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser
Val Thr 675 680 685Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys
Ser Ala Ala Lys 690 695 700Leu Leu Ala Ala Glu Val Lys Asp Lys Lys
Thr Gly Glu Ile Leu Arg705 710 715 720Lys Arg Cys Ala Val His Trp
Val Thr Pro Asp Gly Phe Pro Val Trp 725 730 735Gln Glu Tyr Lys Lys
Pro Ile Gln Thr Arg Leu Asn Leu Met Phe Leu 740 745 750Gly Gln Phe
Arg Leu Gln Pro Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765Ile
Asp Ala His Lys Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770 775
780Ser Gln Asp Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His
Glu785 790 795 800Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp
Ser Phe Gly Thr 805 810 815Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys
Ala Val Arg Glu Thr Met 820 825 830Val Asp Thr Tyr Glu Ser Cys Asp
Val Leu Ala Asp Phe Tyr Asp Gln 835 840 845Phe Ala Asp Gln Leu His
Glu Ser Gln Leu Asp Lys Met Pro Ala Leu 850 855 860Pro Ala Lys Gly
Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880Ala
Phe Ala101883PRTArtificial Sequencechemically synthesized modified
T7 polymerase Y639L/K378R 101Met Asn Thr Ile Asn Ile Ala Lys Asn
Asp Phe Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro Phe Asn Thr Leu
Ala Asp His Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu Gln Leu Ala Leu
Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg Phe Arg Lys Met
Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60Ala Asp Asn Ala Ala
Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys65 70 75 80Met Ile Ala
Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95Gly Lys
Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105
110Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser
115 120 125Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly
Arg Ala 130 135 140Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp
Leu Glu Ala Lys145 150 155 160His Phe Lys Lys Asn Val Glu Glu Gln
Leu Asn Lys Arg Val Gly His 165 170 175Val Tyr Lys Lys Ala Phe Met
Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly
Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205Ser Ile His
Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220Gly
Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230
235 240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala
Thr 245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser Pro Met Phe Gln
Pro Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly
Gly Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro Leu Ala Leu Val
Arg Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg Tyr Glu Asp Val
Tyr Met Pro Glu Val Tyr Lys Ala Ile305 310 315 320Asn Ile Ala Gln
Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335Val Ala
Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345
350Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp
355 360 365Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala
Ala Val 370 375 380Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile
Ser Leu Glu Phe385 390 395 400Met Leu Glu Gln Ala Asn Lys Phe Ala
Asn His Lys Ala Ile Trp Phe 405 410 415Pro Tyr Asn Met Asp Trp Arg
Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn
Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445Gly Lys Pro
Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460Ala
Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470
475 480Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser
Pro 485 490 495Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe
Cys Phe Leu 500 505 510Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His
His Gly Leu Ser Tyr 515 520 525Asn Cys Ser Leu Pro Leu Ala Phe Asp
Gly Ser Cys Ser Gly Ile Gln 530 535 540His Phe Ser Ala Met Leu Arg
Asp Glu Val Gly Gly Arg Ala Val Asn545 550 555 560Leu Leu Pro Ser
Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575Lys Val
Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585
590Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys
595 600 605Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala
Tyr Gly 610 615 620Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr
Leu Ala Leu Gly625 630 635 640Ser Lys Glu Phe Gly Phe Arg Gln Gln
Val Leu Glu Asp Thr Ile Gln 645 650 655Pro Ala Ile Asp Ser Gly Lys
Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met
Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685Val Val Ala
Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700Leu
Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710
715 720Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val
Trp 725 730 735Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu
Met Phe Leu 740 745 750Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr
Asn Lys Asp Ser Glu 755 760 765Ile Asp Ala His Lys Gln Glu Ser Gly
Ile Ala Pro Asn Phe Val His 770 775 780Ser Gln Asp Gly Ser His Leu
Arg Lys Thr Val Val Trp Ala His Glu785 790 795 800Lys Tyr Gly Ile
Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815Ile Pro
Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825
830Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln
835 840 845Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro
Ala Leu 850 855 860Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu
Glu Ser Asp Phe865 870 875 880Ala Phe Ala102883PRTArtificial
Sequencechemically synthesized modified T7 polymerase
P266L/Y639L/H784A 102Met Asn Thr Ile Asn Ile Ala Lys Asn Asp Phe
Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro Phe Asn Thr Leu Ala Asp
His Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu Gln Leu Ala Leu Glu His
Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg Phe Arg Lys Met Phe Glu
Arg Gln Leu Lys Ala Gly Glu Val 50 55 60Ala Asp Asn Ala Ala Ala Lys
Pro Leu Ile Thr Thr Leu Leu Pro Lys65 70 75 80Met Ile Ala Arg Ile
Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95Gly Lys Arg Pro
Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105 110Ala Val
Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120
125Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu
Ala Lys145 150 155 160His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn
Lys Arg Val Gly His 165 170 175Val Tyr Lys Lys Ala Phe Met Gln Val
Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu
Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205Ser Ile His Val Gly
Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220Gly Met Val
Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235
240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser Leu Met Phe Gln Pro
Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly
Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro Leu Ala Leu Val Arg
Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg Tyr Glu Asp Val Tyr
Met Pro Glu Val Tyr Lys Ala Ile305 310 315 320Asn Ile Ala Gln Asn
Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335Val Ala Asn
Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350Pro
Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360
365Met Asn Pro Glu Ala Leu Thr Ala Trp Lys Arg Ala Ala Ala Ala Val
370 375 380Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu
Glu Phe385 390 395 400Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His
Lys Ala Ile Trp Phe 405 410 415Pro Tyr Asn Met Asp Trp Arg Gly Arg
Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met
Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445Gly Lys Pro Ile Gly
Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460Ala Asn Cys
Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475
480Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys
Phe Leu 500 505 510Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His
Gly Leu Ser Tyr 515 520 525Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly
Ser Cys Ser Gly Ile Gln 530 535 540His Phe Ser Ala Met Leu Arg Asp
Glu Val Gly Gly Arg Ala Val Asn545 550 555 560Leu Leu Pro Ser Glu
Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575Lys Val Asn
Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590Glu
Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600
605Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala
Leu Gly625 630 635 640Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu
Glu Asp Thr Ile Gln 645 650 655Pro Ala Ile Asp Ser Gly Lys Gly Leu
Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys
Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685Val Val Ala Ala Val
Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700Leu Leu Ala
Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715
720Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met
Phe Leu 740 745 750Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn
Lys Asp Ser Glu 755 760 765Ile Asp Ala His Lys Gln Glu Ser Gly Ile
Ala Pro Asn Phe Val Ala 770 775 780Ser Gln Asp Gly Ser His Leu Arg
Lys Thr Val Val Trp Ala His Glu785 790 795 800Lys Tyr Gly Ile Glu
Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815Ile Pro Ala
Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830Val
Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840
845Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser
Asp Phe865 870 875 880Ala Phe Ala103883PRTArtificial
Sequencechemically synthesized modified T7 polymerase
P266L/Y639L/H784A/K378R 103Met Asn Thr Ile Asn Ile Ala Lys Asn Asp
Phe Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile Pro Phe Asn Thr Leu Ala
Asp His Tyr Gly Glu Arg Leu 20 25 30Ala Arg Glu Gln Leu Ala Leu Glu
His Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala Arg Phe Arg Lys Met Phe
Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55 60Ala Asp Asn Ala Ala Ala
Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys65 70 75 80Met Ile Ala Arg
Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys Arg 85 90 95Gly Lys Arg
Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys Pro Glu 100 105
110Ala
Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala Cys Leu Thr Ser 115 120
125Ala Asp Asn Thr Thr Val Gln Ala Val Ala Ser Ala Ile Gly Arg Ala
130 135 140Ile Glu Asp Glu Ala Arg Phe Gly Arg Ile Arg Asp Leu Glu
Ala Lys145 150 155 160His Phe Lys Lys Asn Val Glu Glu Gln Leu Asn
Lys Arg Val Gly His 165 170 175Val Tyr Lys Lys Ala Phe Met Gln Val
Val Glu Ala Asp Met Leu Ser 180 185 190Lys Gly Leu Leu Gly Gly Glu
Ala Trp Ser Ser Trp His Lys Glu Asp 195 200 205Ser Ile His Val Gly
Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr 210 215 220Gly Met Val
Ser Leu His Arg Gln Asn Ala Gly Val Val Gly Gln Asp225 230 235
240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala Glu Ala Ile Ala Thr
245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser Leu Met Phe Gln Pro
Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr Gly Ile Thr Gly Gly
Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro Leu Ala Leu Val Arg
Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg Tyr Glu Asp Val Tyr
Met Pro Glu Val Tyr Lys Ala Ile305 310 315 320Asn Ile Ala Gln Asn
Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala 325 330 335Val Ala Asn
Val Ile Thr Lys Trp Lys His Cys Pro Val Glu Asp Ile 340 345 350Pro
Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro Glu Asp Ile Asp 355 360
365Met Asn Pro Glu Ala Leu Thr Ala Trp Arg Arg Ala Ala Ala Ala Val
370 375 380Tyr Arg Lys Asp Lys Ala Arg Lys Ser Arg Arg Ile Ser Leu
Glu Phe385 390 395 400Met Leu Glu Gln Ala Asn Lys Phe Ala Asn His
Lys Ala Ile Trp Phe 405 410 415Pro Tyr Asn Met Asp Trp Arg Gly Arg
Val Tyr Ala Val Ser Met Phe 420 425 430Asn Pro Gln Gly Asn Asp Met
Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440 445Gly Lys Pro Ile Gly
Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly 450 455 460Ala Asn Cys
Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg Ile Lys465 470 475
480Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala Cys Ala Lys Ser Pro
485 490 495Leu Glu Asn Thr Trp Trp Ala Glu Gln Asp Ser Pro Phe Cys
Phe Leu 500 505 510Ala Phe Cys Phe Glu Tyr Ala Gly Val Gln His His
Gly Leu Ser Tyr 515 520 525Asn Cys Ser Leu Pro Leu Ala Phe Asp Gly
Ser Cys Ser Gly Ile Gln 530 535 540His Phe Ser Ala Met Leu Arg Asp
Glu Val Gly Gly Arg Ala Val Asn545 550 555 560Leu Leu Pro Ser Glu
Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys 565 570 575Lys Val Asn
Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr Asp Asn 580 585 590Glu
Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu Ile Ser Glu Lys 595 600
605Val Lys Leu Gly Thr Lys Ala Leu Ala Gly Gln Trp Leu Ala Tyr Gly
610 615 620Val Thr Arg Ser Val Thr Lys Arg Ser Val Met Thr Leu Ala
Leu Gly625 630 635 640Ser Lys Glu Phe Gly Phe Arg Gln Gln Val Leu
Glu Asp Thr Ile Gln 645 650 655Pro Ala Ile Asp Ser Gly Lys Gly Leu
Met Phe Thr Gln Pro Asn Gln 660 665 670Ala Ala Gly Tyr Met Ala Lys
Leu Ile Trp Glu Ser Val Ser Val Thr 675 680 685Val Val Ala Ala Val
Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys 690 695 700Leu Leu Ala
Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile Leu Arg705 710 715
720Lys Arg Cys Ala Val His Trp Val Thr Pro Asp Gly Phe Pro Val Trp
725 730 735Gln Glu Tyr Lys Lys Pro Ile Gln Thr Arg Leu Asn Leu Met
Phe Leu 740 745 750Gly Gln Phe Arg Leu Gln Pro Thr Ile Asn Thr Asn
Lys Asp Ser Glu 755 760 765Ile Asp Ala His Lys Gln Glu Ser Gly Ile
Ala Pro Asn Phe Val Ala 770 775 780Ser Gln Asp Gly Ser His Leu Arg
Lys Thr Val Val Trp Ala His Glu785 790 795 800Lys Tyr Gly Ile Glu
Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr 805 810 815Ile Pro Ala
Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu Thr Met 820 825 830Val
Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp Phe Tyr Asp Gln 835 840
845Phe Ala Asp Gln Leu His Glu Ser Gln Leu Asp Lys Met Pro Ala Leu
850 855 860Pro Ala Lys Gly Asn Leu Asn Leu Arg Asp Ile Leu Glu Ser
Asp Phe865 870 875 880Ala Phe Ala10420DNAArtificial
Sequencechemically synthesized 5'-primer 104agctagctta ctgcatcgac
2010518DNAArtificial Sequencechemically synthesized 3'-primer
105taatacgact cactatag 1810694DNAArtificial Sequencechemically
synthesized control DNA transcription template without leader
sequence ARC2117 106taatacgact cactataggg gagaggagag aacgttctcg
nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn ggatcgttac gactagcatc gatg
9410735DNAArtificial Sequencechemically synthesized sequence
107agtcatgacg ctggctctgg ggtccaaaga gttcg 3510834DNAArtificial
Sequencechemically synthesized sequence 108gaactctttg gaccccagag
ccagcgtcat gact 3410931DNAArtificial Sequencechemically synthesized
sequence 109ggctggcatc tctctgatgt tccaaccttg c 3111031DNAArtificial
Sequencechemically synthesized sequence 110gcaaggttgg aacatcagag
agatgccagc c 3111134DNAArtificial Sequencechemically synthesized
sequence 111cgctcctaac tttgtagcca gccaagacgg tagc
3411234DNAArtificial Sequencechemically synthesized sequence
112gctaccgtct tggctggcta caaagttagg agcg 3411333DNAArtificial
Sequencechemically synthesized sequence 113gctctcaccg cgtggagacg
tgctgccgct gct 3311433DNAArtificial Sequencechemically synthesized
sequence 114agcagcggca gcacgtctcc acgcggtgag agc
3311566DNAArtificial Sequencechemically synthesized 5'-primer
115aaaaaaaaaa aaaaaaaaaa aaaaaaataa tacgactcac tataggggag
tacaataacg 60ttctcg 6611620DNAArtificial Sequencechemically
synthesized 3'-primer 116catcgatgct agtcgtaacg 2011724DNAArtificial
Sequencechemically synthesized DNA transcription template
117ggggnnnnnn nnnnnnnnnn nnnn 2411859DNAArtificial
Sequencechemically synthesized primer 118gatcgatcga tcgatcgatc
taatacgact cactataggg gagtacaata acgttctcg 5911924DNAArtificial
Sequencechemically synthesized primer 119catcgatgct agtcgtaacg atcc
241202652DNAArtificial Sequencechemically synthesized wild-type T7
RNA polymerase sequence 120atgaacacga ttaacatcgc taagaacgac
ttctctgaca tcgaactggc tgctatcccg 60ttcaacactc tggctgacca ttacggtgag
cgtttagctc gcgaacagtt ggcccttgag 120catgagtctt acgagatggg
tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180gctggtgagg
ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag
240atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg
caagcgcccg 300acagccttcc agttcctgca agaaatcaag ccggaagccg
tagcgtacat caccattaag 360accactctgg cttgcctaac cagtgctgac
aatacaaccg ttcaggctgt agcaagcgca 420atcggtcggg ccattgagga
cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480cacttcaaga
aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa
540gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg
tggcgaggcg 600tggtcttcgt ggcataagga agactctatt catgtaggag
tacgctgcat cgagatgctc 660attgagtcaa ccggaatggt tagcttacac
cgccaaaatg ctggcgtagt aggtcaagac 720tctgagacta tcgaactcgc
acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780ctggctggca
tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc
840attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt
gcgtactcac 900agtaagaaag cactgatgcg ctacgaagac gtttacatgc
ctgaggtgta caaagcgatt 960aacattgcgc aaaacaccgc atggaaaatc
aacaagaaag tcctagcggt cgccaacgta 1020atcaccaagt ggaagcattg
tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080ccgatgaaac
cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gaaacgtgct
1140gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag
ccttgagttc 1200atgcttgagc aagccaataa gtttgctaac cataaggcca
tctggttccc ttacaacatg 1260gactggcgcg gtcgtgttta cgctgtgtca
atgttcaacc cgcaaggtaa cgatatgacc 1320aaaggactgc ttacgctggc
gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380aaaatccacg
gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag
1440ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact
ggagaacact 1500tggtgggctg agcaagattc tccgttctgc ttccttgcgt
tctgctttga gtacgctggg 1560gtacagcacc acggcctgag ctataactgc
tcccttccgc tggcgtttga cgggtcttgc 1620tctggcatcc agcacttctc
cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680ttgcttccta
gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag
1740attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt
gaccgatgag 1800aacactggtg aaatctctga gaaagtcaag ctgggcacta
aggcactggc tggtcaatgg 1860ctggcttacg gtgttactcg cagtgtgact
aagcgttcag tcatgacgct ggcttacggg 1920tccaaagagt tcggcttccg
tcaacaagtg ctggaagata ccattcagcc agctattgat 1980tccggcaagg
gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg
2040atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa
ctggcttaag 2100tctgctgcta agctgctggc tgctgaggtc aaagataaga
agactggaga gattcttcgc 2160aagcgttgcg ctgtgcattg ggtaactcct
gatggtttcc ctgtgtggca ggaatacaag 2220aagcctattc agacgcgctt
gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280attaacacca
acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct
2340aactttgtac acagccaaga cggtagccac cttcgtaaga ctgtagtgtg
ggcacacgag 2400aagtacggaa tcgaatcttt tgcactgatt cacgactcct
tcggtaccat tccggctgac 2460gctgcgaacc tgttcaaagc agtgcgcgaa
actatggttg acacatatga gtcttgtgat 2520gtactggctg atttctacga
ccagttcgct gaccagttgc acgagtctca attggacaaa 2580atgccagcac
ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc
2640gcgttcgcgt aa 2652121883PRTArtificial Sequencechemically
synthesized wild-type T7 RNA polymerase sequence 121Met Asn Thr Ile
Asn Ile Ala Lys Asn Asp Phe Ser Asp Ile Glu Leu1 5 10 15Ala Ala Ile
Pro Phe Asn Thr Leu Ala Asp His Tyr Gly Glu Arg Leu 20 25 30Ala Arg
Glu Gln Leu Ala Leu Glu His Glu Ser Tyr Glu Met Gly Glu 35 40 45Ala
Arg Phe Arg Lys Met Phe Glu Arg Gln Leu Lys Ala Gly Glu Val 50 55
60Ala Asp Asn Ala Ala Ala Lys Pro Leu Ile Thr Thr Leu Leu Pro Lys65
70 75 80Met Ile Ala Arg Ile Asn Asp Trp Phe Glu Glu Val Lys Ala Lys
Arg 85 90 95Gly Lys Arg Pro Thr Ala Phe Gln Phe Leu Gln Glu Ile Lys
Pro Glu 100 105 110Ala Val Ala Tyr Ile Thr Ile Lys Thr Thr Leu Ala
Cys Leu Thr Ser 115 120 125Ala Asp Asn Thr Thr Val Gln Ala Val Ala
Ser Ala Ile Gly Arg Ala 130 135 140Ile Glu Asp Glu Ala Arg Phe Gly
Arg Ile Arg Asp Leu Glu Ala Lys145 150 155 160His Phe Lys Lys Asn
Val Glu Glu Gln Leu Asn Lys Arg Val Gly His 165 170 175Val Tyr Lys
Lys Ala Phe Met Gln Val Val Glu Ala Asp Met Leu Ser 180 185 190Lys
Gly Leu Leu Gly Gly Glu Ala Trp Ser Ser Trp His Lys Glu Asp 195 200
205Ser Ile His Val Gly Val Arg Cys Ile Glu Met Leu Ile Glu Ser Thr
210 215 220Gly Met Val Ser Leu His Arg Gln Asn Ala Gly Val Val Gly
Gln Asp225 230 235 240Ser Glu Thr Ile Glu Leu Ala Pro Glu Tyr Ala
Glu Ala Ile Ala Thr 245 250 255Arg Ala Gly Ala Leu Ala Gly Ile Ser
Pro Met Phe Gln Pro Cys Val 260 265 270Val Pro Pro Lys Pro Trp Thr
Gly Ile Thr Gly Gly Gly Tyr Trp Ala 275 280 285Asn Gly Arg Arg Pro
Leu Ala Leu Val Arg Thr His Ser Lys Lys Ala 290 295 300Leu Met Arg
Tyr Glu Asp Val Tyr Met Pro Glu Val Tyr Lys Ala Ile305 310 315
320Asn Ile Ala Gln Asn Thr Ala Trp Lys Ile Asn Lys Lys Val Leu Ala
325 330 335Val Ala Asn Val Ile Thr Lys Trp Lys His Cys Pro Val Glu
Asp Ile 340 345 350Pro Ala Ile Glu Arg Glu Glu Leu Pro Met Lys Pro
Glu Asp Ile Asp 355 360 365Met Asn Pro Glu Ala Leu Thr Ala Trp Lys
Arg Ala Ala Ala Ala Val 370 375 380Tyr Arg Lys Asp Lys Ala Arg Lys
Ser Arg Arg Ile Ser Leu Glu Phe385 390 395 400Met Leu Glu Gln Ala
Asn Lys Phe Ala Asn His Lys Ala Ile Trp Phe 405 410 415Pro Tyr Asn
Met Asp Trp Arg Gly Arg Val Tyr Ala Val Ser Met Phe 420 425 430Asn
Pro Gln Gly Asn Asp Met Thr Lys Gly Leu Leu Thr Leu Ala Lys 435 440
445Gly Lys Pro Ile Gly Lys Glu Gly Tyr Tyr Trp Leu Lys Ile His Gly
450 455 460Ala Asn Cys Ala Gly Val Asp Lys Val Pro Phe Pro Glu Arg
Ile Lys465 470 475 480Phe Ile Glu Glu Asn His Glu Asn Ile Met Ala
Cys Ala Lys Ser Pro 485 490 495Leu Glu Asn Thr Trp Trp Ala Glu Gln
Asp Ser Pro Phe Cys Phe Leu 500 505 510Ala Phe Cys Phe Glu Tyr Ala
Gly Val Gln His His Gly Leu Ser Tyr 515 520 525Asn Cys Ser Leu Pro
Leu Ala Phe Asp Gly Ser Cys Ser Gly Ile Gln 530 535 540His Phe Ser
Ala Met Leu Arg Asp Glu Val Gly Gly Arg Ala Val Asn545 550 555
560Leu Leu Pro Ser Glu Thr Val Gln Asp Ile Tyr Gly Ile Val Ala Lys
565 570 575Lys Val Asn Glu Ile Leu Gln Ala Asp Ala Ile Asn Gly Thr
Asp Asn 580 585 590Glu Val Val Thr Val Thr Asp Glu Asn Thr Gly Glu
Ile Ser Glu Lys 595 600 605Val Lys Leu Gly Thr Lys Ala Leu Ala Gly
Gln Trp Leu Ala Tyr Gly 610 615 620Val Thr Arg Ser Val Thr Lys Arg
Ser Val Met Thr Leu Ala Tyr Gly625 630 635 640Ser Lys Glu Phe Gly
Phe Arg Gln Gln Val Leu Glu Asp Thr Ile Gln 645 650 655Pro Ala Ile
Asp Ser Gly Lys Gly Leu Met Phe Thr Gln Pro Asn Gln 660 665 670Ala
Ala Gly Tyr Met Ala Lys Leu Ile Trp Glu Ser Val Ser Val Thr 675 680
685Val Val Ala Ala Val Glu Ala Met Asn Trp Leu Lys Ser Ala Ala Lys
690 695 700Leu Leu Ala Ala Glu Val Lys Asp Lys Lys Thr Gly Glu Ile
Leu Arg705 710 715 720Lys Arg Cys Ala Val His Trp Val Thr Pro Asp
Gly Phe Pro Val Trp 725 730 735Gln Glu Tyr Lys Lys Pro Ile Gln Thr
Arg Leu Asn Leu Met Phe Leu 740 745 750Gly Gln Phe Arg Leu Gln Pro
Thr Ile Asn Thr Asn Lys Asp Ser Glu 755 760 765Ile Asp Ala His Lys
Gln Glu Ser Gly Ile Ala Pro Asn Phe Val His 770 775 780Ser Gln Asp
Gly Ser His Leu Arg Lys Thr Val Val Trp Ala His Glu785 790 795
800Lys Tyr Gly Ile Glu Ser Phe Ala Leu Ile His Asp Ser Phe Gly Thr
805 810 815Ile Pro Ala Asp Ala Ala Asn Leu Phe Lys Ala Val Arg Glu
Thr Met 820 825 830Val Asp Thr Tyr Glu Ser Cys Asp Val Leu Ala Asp
Phe Tyr Asp Gln 835 840 845Phe Ala Asp Gln Leu His Glu Ser Gln Leu
Asp Lys Met Pro Ala Leu 850 855 860Pro Ala Lys Gly Asn Leu Asn Leu
Arg Asp Ile Leu Glu Ser Asp Phe865 870 875 880Ala Phe
Ala1222652DNAArtificial Sequencechemically synthesized mutant
T7 RNA polymerase Y639L/H784A sequence 122atgaacacga ttaacatcgc
taagaacgac ttctctgaca tcgaactggc tgctatcccg 60ttcaacactc tggctgacca
ttacggtgag cgtttagctc gcgaacagtt ggcccttgag 120catgagtctt
acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa
180gctggtgagg ttgcggataa cgctgccgcc aagcctctca tcactaccct
actccctaag 240atgattgcac gcatcaacga ctggtttgag gaagtgaaag
ctaagcgcgg caagcgcccg 300acagccttcc agttcctgca agaaatcaag
ccggaagccg tagcgtacat caccattaag 360accactctgg cttgcctaac
cagtgctgac aatacaaccg ttcaggctgt agcaagcgca 420atcggtcggg
ccattgagga cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag
480cacttcaaga aaaacgttga ggaacaactc aacaagcgcg tagggcacgt
ctacaagaaa 540gcatttatgc aagttgtcga ggctgacatg ctctctaagg
gtctactcgg tggcgaggcg 600tggtcttcgt ggcataagga agactctatt
catgtaggag tacgctgcat cgagatgctc 660attgagtcaa ccggaatggt
tagcttacac cgccaaaatg ctggcgtagt aggtcaagac 720tctgagacta
tcgaactcgc acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg
780ctggctggca tctctccgat gttccaacct tgcgtagttc ctcctaagcc
gtggactggc 840attactggtg gtggctattg ggctaacggt cgtcgtcctc
tggcgctggt gcgtactcac 900agtaagaaag cactgatgcg ctacgaagac
gtttacatgc ctgaggtgta caaagcgatt 960aacattgcgc aaaacaccgc
atggaaaatc aacaagaaag tcctagcggt cgccaacgta 1020atcaccaagt
ggaagcattg tccggtcgag gacatccctg cgattgagcg tgaagaactc
1080ccgatgaaac cggaagacat cgacatgaat cctgaggctc tcaccgcgtg
gaaacgtgct 1140gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc
gccgtatcag ccttgagttc 1200atgcttgagc aagccaataa gtttgctaac
cataaggcca tctggttccc ttacaacatg 1260gactggcgcg gtcgtgttta
cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc 1320aaaggactgc
ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta ctactggctg
1380aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc cgttccctga
gcgcatcaag 1440ttcattgagg aaaaccacga gaacatcatg gcttgcgcta
agtctccact ggagaacact 1500tggtgggctg agcaagattc tccgttctgc
ttccttgcgt tctgctttga gtacgctggg 1560gtacagcacc acggcctgag
ctataactgc tcccttccgc tggcgtttga cgggtcttgc 1620tctggcatcc
agcacttctc cgcgatgctc cgagatgagg taggtggtcg cgcggttaac
1680ttgcttccta gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa
agtcaacgag 1740attctacaag cagacgcaat caatgggacc gataacgaag
tagttaccgt gaccgatgag 1800aacactggtg aaatctctga gaaagtcaag
ctgggcacta aggcactggc tggtcaatgg 1860ctggcttacg gtgttactcg
cagtgtgact aagcgttcag tcatgacgct ggctctgggg 1920tccaaagagt
tcggcttccg tcaacaagtg ctggaagata ccattcagcc agctattgat
1980tccggcaagg gtctgatgtt cactcagccg aatcaggctg ctggatacat
ggctaagctg 2040atttgggaat ctgtgagcgt gacggtggta gctgcggttg
aagcaatgaa ctggcttaag 2100tctgctgcta agctgctggc tgctgaggtc
aaagataaga agactggaga gattcttcgc 2160aagcgttgcg ctgtgcattg
ggtaactcct gatggtttcc ctgtgtggca ggaatacaag 2220aagcctattc
agacgcgctt gaacctgatg ttcctcggtc agttccgctt acagcctacc
2280attaacacca acaaagatag cgagattgat gcacacaaac aggagtctgg
tatcgctcct 2340aactttgtag ccagccaaga cggtagccac cttcgtaaga
ctgtagtgtg ggcacacgag 2400aagtacggaa tcgaatcttt tgcactgatt
cacgactcct tcggtaccat tccggctgac 2460gctgcgaacc tgttcaaagc
agtgcgcgaa actatggttg acacatatga gtcttgtgat 2520gtactggctg
atttctacga ccagttcgct gaccagttgc acgagtctca attggacaaa
2580atgccagcac ttccggctaa aggtaacttg aacctccgtg acatcttaga
gtcggacttc 2640gcgttcgcgt aa 26521232652DNAArtificial
Sequencechemically synthesized mutant T7 RNA polymerase
Y639L/H784A/K378R sequence 123atgaacacga ttaacatcgc taagaacgac
ttctctgaca tcgaactggc tgctatcccg 60ttcaacactc tggctgacca ttacggtgag
cgtttagctc gcgaacagtt ggcccttgag 120catgagtctt acgagatggg
tgaagcacgc ttccgcaaga tgtttgagcg tcaacttaaa 180gctggtgagg
ttgcggataa cgctgccgcc aagcctctca tcactaccct actccctaag
240atgattgcac gcatcaacga ctggtttgag gaagtgaaag ctaagcgcgg
caagcgcccg 300acagccttcc agttcctgca agaaatcaag ccggaagccg
tagcgtacat caccattaag 360accactctgg cttgcctaac cagtgctgac
aatacaaccg ttcaggctgt agcaagcgca 420atcggtcggg ccattgagga
cgaggctcgc ttcggtcgta tccgtgacct tgaagctaag 480cacttcaaga
aaaacgttga ggaacaactc aacaagcgcg tagggcacgt ctacaagaaa
540gcatttatgc aagttgtcga ggctgacatg ctctctaagg gtctactcgg
tggcgaggcg 600tggtcttcgt ggcataagga agactctatt catgtaggag
tacgctgcat cgagatgctc 660attgagtcaa ccggaatggt tagcttacac
cgccaaaatg ctggcgtagt aggtcaagac 720tctgagacta tcgaactcgc
acctgaatac gctgaggcta tcgcaacccg tgcaggtgcg 780ctggctggca
tctctccgat gttccaacct tgcgtagttc ctcctaagcc gtggactggc
840attactggtg gtggctattg ggctaacggt cgtcgtcctc tggcgctggt
gcgtactcac 900agtaagaaag cactgatgcg ctacgaagac gtttacatgc
ctgaggtgta caaagcgatt 960aacattgcgc aaaacaccgc atggaaaatc
aacaagaaag tcctagcggt cgccaacgta 1020atcaccaagt ggaagcattg
tccggtcgag gacatccctg cgattgagcg tgaagaactc 1080ccgatgaaac
cggaagacat cgacatgaat cctgaggctc tcaccgcgtg gagacgtgct
1140gccgctgctg tgtaccgcaa ggacaaggct cgcaagtctc gccgtatcag
ccttgagttc 1200atgcttgagc aagccaataa gtttgctaac cataaggcca
tctggttccc ttacaacatg 1260gactggcgcg gtcgtgttta cgctgtgtca
atgttcaacc cgcaaggtaa cgatatgacc 1320aaaggactgc ttacgctggc
gaaaggtaaa ccaatcggta aggaaggtta ctactggctg 1380aaaatccacg
gtgcaaactg tgcgggtgtc gataaggttc cgttccctga gcgcatcaag
1440ttcattgagg aaaaccacga gaacatcatg gcttgcgcta agtctccact
ggagaacact 1500tggtgggctg agcaagattc tccgttctgc ttccttgcgt
tctgctttga gtacgctggg 1560gtacagcacc acggcctgag ctataactgc
tcccttccgc tggcgtttga cgggtcttgc 1620tctggcatcc agcacttctc
cgcgatgctc cgagatgagg taggtggtcg cgcggttaac 1680ttgcttccta
gtgaaaccgt tcaggacatc tacgggattg ttgctaagaa agtcaacgag
1740attctacaag cagacgcaat caatgggacc gataacgaag tagttaccgt
gaccgatgag 1800aacactggtg aaatctctga gaaagtcaag ctgggcacta
aggcactggc tggtcaatgg 1860ctggcttacg gtgttactcg cagtgtgact
aagcgttcag tcatgacgct ggctctgggg 1920tccaaagagt tcggcttccg
tcaacaagtg ctggaagata ccattcagcc agctattgat 1980tccggcaagg
gtctgatgtt cactcagccg aatcaggctg ctggatacat ggctaagctg
2040atttgggaat ctgtgagcgt gacggtggta gctgcggttg aagcaatgaa
ctggcttaag 2100tctgctgcta agctgctggc tgctgaggtc aaagataaga
agactggaga gattcttcgc 2160aagcgttgcg ctgtgcattg ggtaactcct
gatggtttcc ctgtgtggca ggaatacaag 2220aagcctattc agacgcgctt
gaacctgatg ttcctcggtc agttccgctt acagcctacc 2280attaacacca
acaaagatag cgagattgat gcacacaaac aggagtctgg tatcgctcct
2340aactttgtag ccagccaaga cggtagccac cttcgtaaga ctgtagtgtg
ggcacacgag 2400aagtacggaa tcgaatcttt tgcactgatt cacgactcct
tcggtaccat tccggctgac 2460gctgcgaacc tgttcaaagc agtgcgcgaa
actatggttg acacatatga gtcttgtgat 2520gtactggctg atttctacga
ccagttcgct gaccagttgc acgagtctca attggacaaa 2580atgccagcac
ttccggctaa aggtaacttg aacctccgtg acatcttaga gtcggacttc
2640gcgttcgcgt aa 26521242652DNAArtificial Sequencechemically
synthesized mutant T7 RNA polymerase P266L/Y639L/H784A sequence
124atgaacacga ttaacatcgc taagaacgac ttctctgaca tcgaactggc
tgctatcccg 60ttcaacactc tggctgacca ttacggtgag cgtttagctc gcgaacagtt
ggcccttgag 120catgagtctt acgagatggg tgaagcacgc ttccgcaaga
tgtttgagcg tcaacttaaa 180gctggtgagg ttgcggataa cgctgccgcc
aagcctctca tcactaccct actccctaag 240atgattgcac gcatcaacga
ctggtttgag gaagtgaaag ctaagcgcgg caagcgcccg 300acagccttcc
agttcctgca agaaatcaag ccggaagccg tagcgtacat caccattaag
360accactctgg cttgcctaac cagtgctgac aatacaaccg ttcaggctgt
agcaagcgca 420atcggtcggg ccattgagga cgaggctcgc ttcggtcgta
tccgtgacct tgaagctaag 480cacttcaaga aaaacgttga ggaacaactc
aacaagcgcg tagggcacgt ctacaagaaa 540gcatttatgc aagttgtcga
ggctgacatg ctctctaagg gtctactcgg tggcgaggcg 600tggtcttcgt
ggcataagga agactctatt catgtaggag tacgctgcat cgagatgctc
660attgagtcaa ccggaatggt tagcttacac cgccaaaatg ctggcgtagt
aggtcaagac 720tctgagacta tcgaactcgc acctgaatac gctgaggcta
tcgcaacccg tgcaggtgcg 780ctggctggca tctctctgat gttccaacct
tgcgtagttc ctcctaagcc gtggactggc 840attactggtg gtggctattg
ggctaacggt cgtcgtcctc tggcgctggt gcgtactcac 900agtaagaaag
cactgatgcg ctacgaagac gtttacatgc ctgaggtgta caaagcgatt
960aacattgcgc aaaacaccgc atggaaaatc aacaagaaag tcctagcggt
cgccaacgta 1020atcaccaagt ggaagcattg tccggtcgag gacatccctg
cgattgagcg tgaagaactc 1080ccgatgaaac cggaagacat cgacatgaat
cctgaggctc tcaccgcgtg gaaacgtgct 1140gccgctgctg tgtaccgcaa
ggacaaggct cgcaagtctc gccgtatcag ccttgagttc 1200atgcttgagc
aagccaataa gtttgctaac cataaggcca tctggttccc ttacaacatg
1260gactggcgcg gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa
cgatatgacc 1320aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta
aggaaggtta ctactggctg 1380aaaatccacg gtgcaaactg tgcgggtgtc
gataaggttc cgttccctga gcgcatcaag 1440ttcattgagg aaaaccacga
gaacatcatg gcttgcgcta agtctccact ggagaacact 1500tggtgggctg
agcaagattc tccgttctgc ttccttgcgt tctgctttga gtacgctggg
1560gtacagcacc acggcctgag ctataactgc tcccttccgc tggcgtttga
cgggtcttgc 1620tctggcatcc agcacttctc cgcgatgctc cgagatgagg
taggtggtcg cgcggttaac 1680ttgcttccta gtgaaaccgt tcaggacatc
tacgggattg ttgctaagaa agtcaacgag 1740attctacaag cagacgcaat
caatgggacc gataacgaag tagttaccgt gaccgatgag 1800aacactggtg
aaatctctga gaaagtcaag ctgggcacta aggcactggc tggtcaatgg
1860ctggcttacg gtgttactcg cagtgtgact aagcgttcag tcatgacgct
ggctctgggg 1920tccaaagagt tcggcttccg tcaacaagtg ctggaagata
ccattcagcc agctattgat 1980tccggcaagg gtctgatgtt cactcagccg
aatcaggctg ctggatacat ggctaagctg 2040atttgggaat ctgtgagcgt
gacggtggta gctgcggttg aagcaatgaa ctggcttaag 2100tctgctgcta
agctgctggc tgctgaggtc aaagataaga agactggaga gattcttcgc
2160aagcgttgcg ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca
ggaatacaag 2220aagcctattc agacgcgctt gaacctgatg ttcctcggtc
agttccgctt acagcctacc 2280attaacacca acaaagatag cgagattgat
gcacacaaac aggagtctgg tatcgctcct 2340aactttgtag ccagccaaga
cggtagccac cttcgtaaga ctgtagtgtg ggcacacgag 2400aagtacggaa
tcgaatcttt tgcactgatt cacgactcct tcggtaccat tccggctgac
2460gctgcgaacc tgttcaaagc agtgcgcgaa actatggttg acacatatga
gtcttgtgat 2520gtactggctg atttctacga ccagttcgct gaccagttgc
acgagtctca attggacaaa 2580atgccagcac ttccggctaa aggtaacttg
aacctccgtg acatcttaga gtcggacttc 2640gcgttcgcgt aa
26521252652DNAArtificial Sequencechemically synthesized mutant T7
RNA polymerase P266L/Y639L/H784A/K378R sequence 125atgaacacga
ttaacatcgc taagaacgac ttctctgaca tcgaactggc tgctatcccg 60ttcaacactc
tggctgacca ttacggtgag cgtttagctc gcgaacagtt ggcccttgag
120catgagtctt acgagatggg tgaagcacgc ttccgcaaga tgtttgagcg
tcaacttaaa 180gctggtgagg ttgcggataa cgctgccgcc aagcctctca
tcactaccct actccctaag 240atgattgcac gcatcaacga ctggtttgag
gaagtgaaag ctaagcgcgg caagcgcccg 300acagccttcc agttcctgca
agaaatcaag ccggaagccg tagcgtacat caccattaag 360accactctgg
cttgcctaac cagtgctgac aatacaaccg ttcaggctgt agcaagcgca
420atcggtcggg ccattgagga cgaggctcgc ttcggtcgta tccgtgacct
tgaagctaag 480cacttcaaga aaaacgttga ggaacaactc aacaagcgcg
tagggcacgt ctacaagaaa 540gcatttatgc aagttgtcga ggctgacatg
ctctctaagg gtctactcgg tggcgaggcg 600tggtcttcgt ggcataagga
agactctatt catgtaggag tacgctgcat cgagatgctc 660attgagtcaa
ccggaatggt tagcttacac cgccaaaatg ctggcgtagt aggtcaagac
720tctgagacta tcgaactcgc acctgaatac gctgaggcta tcgcaacccg
tgcaggtgcg 780ctggctggca tctctctgat gttccaacct tgcgtagttc
ctcctaagcc gtggactggc 840attactggtg gtggctattg ggctaacggt
cgtcgtcctc tggcgctggt gcgtactcac 900agtaagaaag cactgatgcg
ctacgaagac gtttacatgc ctgaggtgta caaagcgatt 960aacattgcgc
aaaacaccgc atggaaaatc aacaagaaag tcctagcggt cgccaacgta
1020atcaccaagt ggaagcattg tccggtcgag gacatccctg cgattgagcg
tgaagaactc 1080ccgatgaaac cggaagacat cgacatgaat cctgaggctc
tcaccgcgtg gagacgtgct 1140gccgctgctg tgtaccgcaa ggacaaggct
cgcaagtctc gccgtatcag ccttgagttc 1200atgcttgagc aagccaataa
gtttgctaac cataaggcca tctggttccc ttacaacatg 1260gactggcgcg
gtcgtgttta cgctgtgtca atgttcaacc cgcaaggtaa cgatatgacc
1320aaaggactgc ttacgctggc gaaaggtaaa ccaatcggta aggaaggtta
ctactggctg 1380aaaatccacg gtgcaaactg tgcgggtgtc gataaggttc
cgttccctga gcgcatcaag 1440ttcattgagg aaaaccacga gaacatcatg
gcttgcgcta agtctccact ggagaacact 1500tggtgggctg agcaagattc
tccgttctgc ttccttgcgt tctgctttga gtacgctggg 1560gtacagcacc
acggcctgag ctataactgc tcccttccgc tggcgtttga cgggtcttgc
1620tctggcatcc agcacttctc cgcgatgctc cgagatgagg taggtggtcg
cgcggttaac 1680ttgcttccta gtgaaaccgt tcaggacatc tacgggattg
ttgctaagaa agtcaacgag 1740attctacaag cagacgcaat caatgggacc
gataacgaag tagttaccgt gaccgatgag 1800aacactggtg aaatctctga
gaaagtcaag ctgggcacta aggcactggc tggtcaatgg 1860ctggcttacg
gtgttactcg cagtgtgact aagcgttcag tcatgacgct ggctctgggg
1920tccaaagagt tcggcttccg tcaacaagtg ctggaagata ccattcagcc
agctattgat 1980tccggcaagg gtctgatgtt cactcagccg aatcaggctg
ctggatacat ggctaagctg 2040atttgggaat ctgtgagcgt gacggtggta
gctgcggttg aagcaatgaa ctggcttaag 2100tctgctgcta agctgctggc
tgctgaggtc aaagataaga agactggaga gattcttcgc 2160aagcgttgcg
ctgtgcattg ggtaactcct gatggtttcc ctgtgtggca ggaatacaag
2220aagcctattc agacgcgctt gaacctgatg ttcctcggtc agttccgctt
acagcctacc 2280attaacacca acaaagatag cgagattgat gcacacaaac
aggagtctgg tatcgctcct 2340aactttgtag ccagccaaga cggtagccac
cttcgtaaga ctgtagtgtg ggcacacgag 2400aagtacggaa tcgaatcttt
tgcactgatt cacgactcct tcggtaccat tccggctgac 2460gctgcgaacc
tgttcaaagc agtgcgcgaa actatggttg acacatatga gtcttgtgat
2520gtactggctg atttctacga ccagttcgct gaccagttgc acgagtctca
attggacaaa 2580atgccagcac ttccggctaa aggtaacttg aacctccgtg
acatcttaga gtcggacttc 2640gcgttcgcgt aa 265212689DNAArtificial
Sequencechemically synthesized DNA transcription template
126gggagaattc cgaccagaag cttnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60nnncatatgt gcgtctacat ggatcctca 8912789DNAArtificial
Sequencechemically synthesized DNA transcription template
127gggagagcgg aagccgtgct ggggccnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60nnnnnncata acccagaggt cgatggatc 8912894DNAArtificial
Sequencechemically synthesized DNA transcription template
128gggagagaca agcttgggtc nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 60agaagagaaa gagaagttaa ttaaggatcc tcag
9412986DNAArtificial Sequencechemically synthesized DNA
transcription template 129gggagaattc cgaccacaag nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60catatgtgcg tctacatgga tcctca
8613040DNAArtificial Sequencechemically synthesized 5' primer
sequence 130taatacgact cactataggg agaattccga ccagaagctt
4013126DNAArtificial Sequencechemically synthesized 3' primer
sequence 131tgaggatcca tgtagacgca catatg 2613223DNAArtificial
Sequencechemically synthesized 3' primer sequence 132gatccatcga
cctctgggtt atg 2313337DNAArtificial Sequencechemically synthesized
5' primer sequence 133taatacgact cactataggg agagacaagc ttgggtc
3713434DNAArtificial Sequencechemically synthesized 3' primer
sequence 134ctgaggatcc ttaattaact tctctttctc ttct
3413537DNAArtificial Sequencechemically synthesized 5' primer
sequence 135taatacgact cactataggg agaattccga ccacaag
3713610DNAArtificial Sequencechemically synthesized
immunostimulatory motif 136aacgttcgag 1013776DNAArtificial
Sequencechemically synthesized DNA template ARC3428 137gggagacaag
aataaagcga gttnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnaagagtc 60gatgatgctt
agctag 7613877DNAArtificial Sequencechemically synthesized DNA
transcription template 138gggccttgta gcgtgcattc ttgnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnctaacat 60actccgaatc tgtcgaa
7713973DNAArtificial Sequencechemically synthesized DNA
transcription template 139ggagccttcc tccggannnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnntccg 60gtttcccgag ctt
7314087DNAArtificial Sequencechemically synthesized DNA
transcription template 140gggagacaag aataaacgct caannnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnttcgaca ggaggctcac aacaggc
8714124DNAArtificial Sequencechemically synthesized 3' primer
sequence 141ttcgacagat tcggagtatg ttag 2414233DNAArtificial
Sequencechemically synthesized 5' primer sequence 142taatacgact
cactatagga gccttcctcc gga 3314317DNAArtificial Sequencechemically
synthesized 3' primer sequence 143aagctcggga aaccgga
1714440DNAArtificial Sequencechemically synthesized 5' primer
sequence 144taatacgact cactataggg agacaagaat aaacgctcaa
4014524DNAArtificial Sequencechemically synthesized 3' primer
sequence 145gcctgttgtg agcctcctgt cgaa 2414639DNAArtificial
Sequencechemically synthesized 5' primer sequence 146taatacgact
cactataggg gagtacaata accagacat 3914724DNAArtificial
Sequencechemically synthesized 3' primer sequence 147catcgatgct
agtcgtaacg atcc 2414826DNAArtificial Sequencechemically synthesized
3' primer sequence 148tgaggatcca tgtagacgca catatg
2614943DNAArtificial Sequencechemically synthesized 5' primer
sequence 149taatacgact cactataggg agagcggaag ccgtgctggg gcc
4315076DNAArtificial Sequencechemically synthesized DNA
transcription template 150ggggagtaca ataaccagac atnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnggatcgtt 60acgactagca tcgatg
7615140DNAArtificial Sequencechemically synthesized 5' primer
sequence 151taatacgact cactataggg ccttgtagcg tgcattcttg 40
* * * * *